Substituted aliphanes, cyclophanes, heteraphanes, heterophanes, hetero-heteraphanes and metallocenes useful for treating HCV infections

ABSTRACT

The present disclosure provides substituted aliphanes, cyclophanes, heteraphanes, heterophanes, hetero-heteraphanes and metallocenes, of Formula I
 
D-M-D  (Formula I)
 
useful as antiviral agents. In certain embodiments disclosed herein M is a group —P-A-P— where A is
 
                         
Certain substituted aliphanes, cyclophanes, heteraphanes, heterophanes, hetero-heteraphanes and metallocenes disclosed herein are potent and/or selective inhibitors of viral replication, particularly Hepatitis C virus replication. Pharmaceutical compositions/and combinations containing one or more substituted aliphanes, cyclophanes, heteraphanes, heterophanes, hetero-heteraphanes and metallocenes and a pharmaceutically acceptable carrier are also provided by this disclosure. Methods for treating viral infections, including Hepatitis C viral infections are provided by the disclosure.

PRIORITY INFORMATION

This application is a continuation of U.S. application Ser. No.14/328,312, which is a continuation of U.S. application Ser. No.13/482,558, which claims priority from U.S. Provisional Application Nos.61/490,881 filed May 27, 2011, 61/504,905, filed Jul. 6, 2011, and61/567,216, filed Dec. 6, 2011 all of which are hereby incorporated byreference in their entirety.

FIELD OF THE DISCLOSURE

The present disclosure provides substituted aliphanes, cyclophanes,heteraphanes, heterophanes, hetero-heteraphanes and metallocenes, usefulas antiviral agents. Certain substituted aliphanes, cyclophanes,heteraphanes, heterophanes, hetero-heteraphanes and metallocenesdisclosed herein are potent and/or selective inhibitors of viralreplication, particularly Hepatitis C virus replication. Pharmaceuticalcompositions/and combinations containing one or more substitutedaliphanes, cyclophanes, heteraphanes, heterophanes, hetero-heteraphanesand metallocenes and a pharmaceutically acceptable carrier are alsoprovided by this disclosure. Methods for treating viral infections,including Hepatitis C viral infections are provided by the disclosure.

BACKGROUND

An estimated 3% of the world's population is infected with the hepatitisC virus. Of those exposed to HCV, 80% to 85% become chronicallyinfected, at least 30% develop cirrhosis of the liver and 1-4% develophepatocellular carcinoma. Hepatitis C Virus (HCV) is one of the mostprevalent causes of chronic liver disease in the United States,reportedly accounting for about 15 percent of acute viral hepatitis, 60to 70 percent of chronic hepatitis, and up to 50 percent of cirrhosis,end-stage liver disease, and liver cancer. Chronic HCV infection is themost common cause of liver transplantation in the U.S., Australia, andmost of Europe. Hepatitis C causes an estimated 10,000 to 12,000 deathsannually in the United States. While the acute phase of HCV infection isusually associated with mild symptoms, some evidence suggests that onlyabout 15% to 20% of infected people will spontaneously clear HCV.

HCV is an enveloped, single-stranded RNA virus that contains apositive-stranded genome of about 9.6 kb. HCV is classified as a memberof the Hepacivirus genus of the family Flaviviridae. At least 4 strainsof HCV, GT-1-GT-4, have been characterized.

The HCV lifecycle includes entry into host cells; translation of the HCVgenome, polyprotein processing, and replicase complex assembly; RNAreplication, and virion assembly and release. Translation of the HCV RNAgenome yields a more than 3000 amino acid long polyprotein that isprocessed by at least two cellular and two viral proteases. The HCVpolyprotein is:NH2-C-E1-E2-p7-NS2-NS3-NS4A-NS4B-NS5A-NS5B-COOH.

The cellular signal peptidase and signal peptide peptidase have beenreported to be responsible for cleavage of the N-terminal third of thepolyprotein (C-E1-E2-p7) from the nonstructural proteins(NS2-NS3-NS4A-NS4B-NS5A-NS5B). The NS2-NS3 protease mediates a first ciscleavage at the NS2-NS3 site. The NS3-NS4A protease then mediates asecond cis-cleavage at the NS3-NS4A junction. The NS3-NS4A complex thencleaves at three downstream sites to separate the remainingnonstructural proteins. Accurate processing of the polyprotein isasserted to be essential for forming an active HCV replicase complex.

Once the polyprotein has been cleaved, the replicase complex comprisingat least the NS3-NS5B nonstructural proteins assembles. The replicasecomplex is cytoplasmic and membrane-associated. Major enzymaticactivities in the replicase complex include serine protease activity andNTPase helicase activity in NS3, and RNA-dependent RNA polymeraseactivity of NS5B. In the RNA replication process, a complementarynegative strand copy of the genomic RNA is produced. The negative strandcopy is used as a template to synthesize additional positive strandgenomic RNAs that may participate in translation, replication,packaging, or any combination thereof to produce progeny virus. Assemblyof a functional replicase complex has been described as a component ofthe HCV replication mechanism. U.S. Provisional Application No.60/669,872 “Pharmaceutical Compositions and Methods of Inhibiting HCVReplication” filed Apr. 11, 2005, is hereby incorporated by reference inits entirety for its disclosure related to assembly of the replicasecomplex.

Current treatment of hepatitis C infection typically includesadministration of an interferon, such as pegylated interferon (IFN), incombination with ribavirin. The success of current therapies as measuredby sustained virologic response (SVR) depends on the strain of HCV withwhich the patient is infected and the patient's adherence to thetreatment regimen. Only 50% of patients infected with HCV strain GT-1exhibit a sustained virological response. Direct acting antiviral agentssuch as ACH-1625, telaprevir, BMS-790052, and BMS-650032 are in clinicaldevelopment for treatment of chronic HCV. Due to lack of effectivetherapies for treatment for certain HCV strains and the high mutationrate of HCV, new therapies are needed. The present disclosure fulfillsthis need and provides additional advantages, which are describedherein.

SUMMARY

Substituted aliphanes, cyclophanes, heteraphanes, heterophanes,hetero-heteraphanes and metallocenes of Formula I are provided herein.The compounds of Formula I provided in this disclosure posses antiviralactivity.

The disclosure provides compounds of Formula I that are potent and/orselective inhibitors of Hepatitis C virus replication. Without beingbound to any particular theory it is believed the present compounds arepotent and selective inhibitors of HCV NS5a. Pharmaceutical compositionscontaining one or more compounds of Formula I, or a salt of suchcompounds, and one or more pharmaceutically acceptable carriers are alsoprovided herein. Pharmaceutical combinations containing one or morecompounds of Formula I, or a salt of such compounds, at least oneadditional active agent, and one or more pharmaceutically acceptablecarriers are also provided herein.

Also disclosed are methods of treating patients suffering from certainviral infections, particularly HCV infections, by providing to suchpatients an amount of a compound of Formula I effective to reduce signsor symptoms of the viral infection. Methods of treatment includeproviding a compound of Formula I as a single active agent or providinga compound of Formula I in combination with one or more othertherapeutic active agents.

In a first aspect the disclosure includes compounds of Formula ID-M-D  (Formula I)or a pharmaceutically acceptable salt thereof.

Within Formula I, the variables D and M carry the following definitions.

D is T-R— where M is covalently bound to R.

M is —P-A-P—.

P is -J-W— where J is covalently bound to R and W is covalently bound toA, or

P is -J- where J is covalently bound to R and A.

J is independently chosen at each occurrence and is J′ where i is aninteger from 1 to 2.

T is independently chosen at each occurrence and is T^(k) where k is aninteger from 1 to 2; T¹ is —Y—Z, where Y is covalently bound to R and Yis a bond, C₁-C₄alkylene optionally substituted with oxo; and Z is a 5or 6-membered heterocyclic group, each of which T¹ is substituted with(i) at least one substituent selected from —(C═O)OH, —(C═O)NH₂, —(C═O)H,—C₁-C₄alkoxy, C₂-C₄alkanoyl, C₁-C₄alkylester, C₁-C₄alkenylester, mono-or di-C₁-C₄alkylcarboxamide and (ii) optionally substituted with one ormore substituents independently chosen from halogen, hydroxyl,C₁-C₂alkyl, and C₁-C₂alkoxy; and T² is independently chosen at eachoccurrence from C₂-C₆alkanoyl, C₁-C₆alkylester, C₁-C₆alkenylester,C₁-C₆alkylsulfonamide, C₁-C₆alkylsulfonyl, C₂-C₆alkanoyl substitutedwith mono- or di-C₁-C₆hydrocarbylcarbamate, C₂-C₆alkanoyl substitutedwith urea or mono- or di-C₁-C₆alkylurea, and C₂-C₆alkanoyl substitutedwith mono- or di-C₁-C₆alkylcarboxamide, each of which T is optionallysubstituted with 1 or more substituents independently chosen from amino,cyano, hydroxyl, halogen, (C₁-C₄alkoxy)C₀-C₄alkyl, (mono- anddi-C₁-C₄alkylamino)C₀-C₄alkyl, C₁-C₆alkyl, (C₁-C₄thioalkyl)C₀-C₄alkyl,C₃-C₇cycloalkyl, phenyl, C₁-C₄haloalkyl, and C₁-C₄haloalkoxy;

R is independently chosen at each occurrence from

(a) 4- to 6-membered rings containing one or two nitrogen atoms withremaining ring atoms being carbon, which R is saturated or contains 1unsaturated bond and is optionally bridged with an methylene or ethylenebridge, fused to a phenyl or 5- to 6-membered heteroaryl ring; and

(b) 6- to 10-membered fused or spiro bicyclic ring systems containingone or two nitrogen atoms with remaining ring atoms being carbon, which6- to 10-membered bicyclic ring is saturated or contains 1 unsaturatedbond; each R is optionally substituted with one or more substituentsindependently chosen from cyano, hydroxyl, halogen, C₁-C₂alkyl,C₁-C₂alkoxy, C₁-C₂haloalkyl, C₁-C₂haloalkyl, C₁-C₂haloalkylene, andC₁-C₂alkylsulfonyl.

J¹ is phenyl or a 5- to 6-membered heteroaryl group containing 1 to 3heteroatoms independently chosen from N, O, and S, where each J¹ isoptionally substituted with one or more substituents independentlychosen from amino, cyano, hydroxyl, halogen, C₁-C₄alkyl, C₁-C₄alkoxy,mono- and di-C₁-C₄alkylamino, C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

J² is an 8- to 10-membered heteroaryl group containing 1 to 4heteroatoms independently chosen from N, O, and S, wherein J² isoptionally substituted with one or more substituents independentlychosen from amino, cyano, hydroxyl, halogen, C₁-C₄alkyl, C₁-C₄alkoxy,mono- and di-C₁-C₄alkylamino, C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

W is independently chosen at each occurrence and is a phenyl, pyridyl oralkynyl group, optionally substituted with one or more substituentsindependently chosen from amino, cyano, hydroxyl, halogen, C₁-C₄alkyl,C₁-C₄alkoxy, mono- and di-C₁-C₄alkylamino, C₁-C₂haloalkyl, andC₁-C₂haloalkoxy.

A is [j.k]-cyclophane, [j.k]-hetera-phane, [j.k]-hetero-phane,[j.k]-hetero-hetera-phane, or [j.k]-aliphane; where j is an integer from1 to 4, k is an integer from 0 to 4, the difference between j and k isnot more than 2, and each j and k linker optionally contains aheteroatom selected from N, O, and S, and is optionally substituted with1 oxo group, and one or more substituents independently chosen fromhalogen, hydroxy, amino, C₁-C₂alkyl, and C₁-C₂alkoxy; or

A is a [j.k.j′.k′]-cyclophane, where j, j′, k, and k′ are integers from1 to 4, the difference between j and k or k′ is not more than 2, thedifference between j′ and k or k′ is not more than 2, and each j, j′, k,and k′ linker optionally contains a heteroatom selected from N, O, andS, and is optionally substituted with 1 oxo group, and one or moresubstituents independently chosen from halogen, hydroxy, amino,C₁-C₂alkyl, and C₁-C₂alkoxy;

or

A is a group of the formula

wherein Q is a neutral or cationic metal, each of which A is optionallysubstituted with one or more substituents independently chosen fromhalogen, C₁-C₂alkyl, and C₁-C₂alkoxy. In certain embodiments Q is chosenfrom Fe, Co, Cr, Ni, V, Li, Rb, and K; or

A is a group of the formula

which A is optionally substituted with one or more substituentsindependently chosen from halogen, C₁-C₂alkyl, and C₁-C₂alkoxy.

Pharmaceutical compositions and combinations containing a compound ofFormula I, together with a pharmaceutically acceptable carrier areprovided by the disclosure. The compositions and combinations providedby this disclosure may include a compound of Formula I as the onlyactive agent or may include one ore more additional active agents. Incertain embodiments the additional active agent is an NS3a proteaseinhibitor. This disclosure also includes a method of treating hepatitisC infection in a patient, comprising providing a therapeuticallyeffective amount of one or more compounds of Formula I to the patient.The compound of Formula I may be provided as the only active agent ormay be provided together with one or more additional active agents suchas an NS3a protease inhibitor.

Certain compounds of Formula I disclosed herein exhibit good activity inan HCV replication assay, such as the HCV replicon assay set forth inExample 9, which follows. Preferred compounds of Formula I exhibit anEC₅₀ of about 10 micromolar or less, or more preferably an EC₅₀ of about1 micromolar or less; or still more preferably an EC₅₀ of about 100nanomolar or less in an HCV replicon replication assay.

DETAILED DESCRIPTION

Chemical Description and Terminology

Prior to setting forth the invention in detail, it may be helpful toprovide definitions of certain terms to be used in this disclosure.Compounds are described using standard nomenclature. Unless definedotherwise, all technical and scientific terms used herein have the samemeaning as is commonly understood by one of skill in the art to whichthis invention belongs. Unless clearly contraindicated by the contexteach compound name includes the free acid or free base form of thecompound as well as all pharmaceutically acceptable salts of thecompound.

The term “compounds of Formula I” encompasses all compounds that satisfyFormula I, including any enantiomers, racemates and stereoisomers, aswell as all pharmaceutically acceptable salts of such compounds. Thephrase “a compound of Formula I” includes all subgeneric groups ofFormula I, and also includes pharmaceutically acceptable salts of acompound of Formula I, unless clearly contraindicated by the context inwhich this phrase is used.

“Formula II” encompasses all compounds that satisfy Formula II,including any enantiomers, racemates and stereoisomers, as well as allpharmaceutically acceptable salts of such compounds. The phrase “acompound of Formula II” includes all sub generic groups of Formula II,and also includes pharmaceutically acceptable salts of a compound ofFormula II, unless clearly contraindicated by the context in which thisphrase is used.

The terms “a” and “a” do not denote a limitation of quantity, but ratherdenote the presence of at least one of the referenced item. The term“or” means “and/or”. The open-ended transitional phrase “comprising”encompasses the intermediate transitional phrase “consisting essentiallyof” and the close-ended phrase “consisting of.” Claims reciting one ofthese three transitional phrases, or with an alternate transitionalphrase such as “containing” or “including” can be written with any othertransitional phrase unless clearly precluded by the context or art.Recitation of ranges of values are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. The endpoints of all ranges are includedwithin the range and independently combinable. All methods describedherein can be performed in a suitable order unless otherwise indicatedherein or otherwise clearly contradicted by context. The use of any andall examples, or exemplary language (e.g., “such as”), is intendedmerely to better illustrate the invention and does not pose a limitationon the scope of the invention unless otherwise claimed. No language inthe specification should be construed as indicating any non-claimedelement as essential to the practice of the invention as used herein.Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as is commonly understood by one of skill in theart to which this invention belongs.

An “active agent” means a compound (including a compound disclosedherein), element, or mixture that when administered to a patient, aloneor in combination with another compound, element, or mixture, confers,directly or indirectly, a physiological effect on the patient. Theindirect physiological effect may occur via a metabolite or otherindirect mechanism.

A dash (“-”) that is not between two letters or symbols is used toindicate a point of attachment for a substituent. For example, —(C═O)NH2is attached through carbon of the keto (C═O) group.

An “aliphane” as used herein is a group composed of one or twocycloalkyl rings, with at least one aliphatic bridge between twonon-adjacent positions of the single cycloalkyl ring or between the twocycloalkyl rings. The aliphatic bridges contain between 1 and 4 carbonatoms. The aliphatic bridges are optionally substituted with an oxogroup.

“Alkenyl” is an alkyl group as defined herein, covalently bound to thegroup it substitutes by a keto (—(C═O)—) bridge. Alkanoyl groups havethe indicated number of carbon atoms, with the carbon of the keto groupbeing included in the numbered carbon atoms. For example a C₂alkanoylgroup is an acetyl group having the formula CH₃(C═O)—.

“Alkyl” is a branched or straight chain saturated aliphatic hydrocarbongroup, having the specified number of carbon atoms, generally from 1 toabout 12 carbon atoms. The term C₁-C₆alkyl as used herein indicates analkyl group having from 1, 2, 3, 4, 5, or 6 carbon atoms. Otherembodiments include alkyl groups having from 1 to 8 carbon atoms, 1 to 4carbon atoms or 1 or 2 carbon atoms, e.g. C₁-C₈alkyl, C₁-C₄alkyl, andC₁-C₂alkyl. When C₀-C_(n) alkyl is used herein in conjunction withanother group, for example, (C₁-C₄alkoxy)C₀-C₄ alkyl, the indicatedgroup, in this case alkoxy, is either directly bound by a singlecovalent bond (C₀alkyl), or attached by an alkyl chain having thespecified number of carbon atoms, in this case 1, 2, 3, or 4 carbonatoms. Examples of alkyl include, but are not limited to, methyl, ethyl,n-propyl, isopropyl, n-butyl, 3-methylbutyl, t-butyl, n-pentyl, andsec-pentyl.

“Alkynyl” is a branched or straight chain aliphatic hydrocarbon grouphaving one or more triple carbon-carbon bonds that may occur at anystable point along the chain, having the specified number of carbonatoms. Examples of alkynyl include, but are not limited to, ethynyl andpropynyl.

“Alkoxy” is an alkyl group as defined above with the indicated number ofcarbon atoms covalently bound to the group it substitutes by an oxygenbridge (—O—). Examples of alkoxy include, but are not limited to,methoxy, ethoxy, n-propoxy, i-propoxy, n-butoxy, 2-butoxy, t-butoxy,n-pentoxy, 2-pentoxy, 3-pentoxy, isopentoxy, neopentoxy, n-hexoxy,2-hexoxy, 3-hexoxy, and 3-methylpentoxy.

The term “alkylester” indicates an alkyl group as defined hereincovalently bound to the group it substitutes by an ester linkage. Theester linkage may be in either orientation, e.g., a group of the formula—O(C═O)alkyl or a group of the formula —(C═O)Oalkyl.

“Alkylsulfonyl” is a group of the formula —SO₂alkyl, where the alkylgroup carries the definition set forth herein.

“Cycloalkyl” is a saturated hydrocarbon ring group, having the specifiednumber of carbon atoms. Monocyclic cycloalkyl groups typically have from3 to about 8 carbon ring atoms or from 3 to 7 (3, 4, 5, 6, or 7) carbonring atoms. Cycloalkyl substituents may be pendant from a substitutednitrogen or carbon atom, or a substituted carbon atom that may have twosubstituents may have a cycloalkyl group, which is attached as a spirogroup. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl,cyclopentyl, and cyclohexyl.

A “cyclophane” as used herein is a group composed of one or two aromaticrings, usually benzene rings, with at least one aliphatic bridge betweentwo non-adjacent positions of the single aromatic ring or between thetwo aromatic rings. The aliphatic bridges are in the meta or para ormeta, para (meta on one aromatic ring and para on the other) orientationand contain between 1 and 4 carbon atoms. The aliphatic bridges areoptionally substituted with an oxo group.

“Haloalkyl” indicates both branched and straight-chain alkyl groupshaving the specified number of carbon atoms, substituted with 1 or morehalogen atoms, up to the maximum allowable number of halogen atoms.Examples of haloalkyl include, but are not limited to, trifluoromethyl,difluoromethyl, 2-fluoroethyl, and penta-fluoroethyl.

“Haloalkoxy” indicates a haloalkyl group as defined herein attachedthrough an oxygen bridge (oxygen of an alcohol radical).

“Halo” or “halogen” indicates any of fluoro, chloro, bromo, and iodo.

“Heteroaryl” indicates a stable monocyclic aromatic ring having theindicated number of ring atoms which contains from 1 to 3, or in someembodiments from 1 to 2, heteroatoms chosen from N, O, and S, withremaining ring atoms being carbon, or a stable bicyclic or tricyclicsystem containing at least one 5- to 7-membered aromatic ring whichcontains from 1 to 3, or in some embodiments from 1 to 2, heteroatomschosen from N, O, and S, with remaining ring atoms being carbon.Monocyclic heteroaryl groups typically have from 5 to 7 ring atoms. Insome embodiments bicyclic heteroaryl groups are 9- to 10-memberedheteroaryl groups, that is, groups containing 9 or 10 ring atoms inwhich one 5- to 7-member aromatic ring is fused to a second aromatic ornon-aromatic ring. When the total number of S and O atoms in theheteroaryl group exceeds 1, these heteroatoms are not adjacent to oneanother. It is preferred that the total number of S and O atoms in theheteroaryl group is not more than 2. It is particularly preferred thatthe total number of S and O atoms in the aromatic heterocycle is notmore than 1. Examples of heteroaryl groups include, but are not limitedto, oxazolyl, pyranyl, pyrazinyl, pyrazolopyrimidinyl, pyrazolyl,pyridizinyl, pyridyl, pyrimidinyl, pyrrolyl, quinolinyl, tetrazolyl,thiazolyl, thienylpyrazolyl, thiophenyl, triazolyl, benzo[d]oxazolyl,benzofuranyl, benzothiazolyl, benzothiophenyl, benzoxadiazolyl,dihydrobenzodioxynyl, furanyl, imidazolyl, indolyl, and isoxazolyl.“Heteroaryloxy” is a heteroaryl group as described bound to the group itsubstituted via an oxygen bridge.

A “hetera-phane” as used herein is a group composed of one or twoaromatic rings, usually benzene rings, with at least one aliphaticbridge between two non-adjacent positions of the single aromatic ring orbetween the two aromatic rings that contains a heteroatom. The aliphaticbridges are in the meta or para or meta, para (meta on one aromatic ringand para on the other) orientation and contain between 1 and 4 atoms, atleast one of which is a heteroatom with the remaining atoms being carbonatoms. The aliphatic bridges are optionally substituted with an oxogroup.

A “hetero-phane” as used herein is a group composed of one or twoaromatic rings, wherein at least one aromatic ring is heteroaryl, withat least one aliphatic bridge between two non-adjacent positions of thesingle aromatic ring or between the two aromatic rings. The aliphaticbridges are in the meta or para or meta, para (meta on one aromatic ringand para on the other) orientation and contain between 1 and 4 carbonatoms. The aliphatic bridges are optionally substituted with an oxogroup.

A “hetero-hetera-phane” as used herein is a group composed of one or twoaromatic rings, wherein at least one aromatic ring is heteroaryl, withat least one aliphatic bridge between two non-adjacent positions of thesingle aromatic ring or between the two aromatic rings that contains ahetero atom chosen from N, O, or S, and one aliphatic bridge between twonon-adjacent positions of the single aromatic ring or between the twoaromatic rings that does not contain a heteroatom. The aliphatic bridgesare in the meta or para or meta, para (meta on one aromatic ring andpara on the other) orientation and contain between 1 and 4 atoms, atleast one of which is a heteroatom with the remaining atoms being carbonatoms. The aliphatic bridges are optionally substituted with an oxogroup.

“Hydrocarbyl” is a saturated or unsaturated aliphatic group containingthe indicated number of carbon atoms. “Hydrocarbyl may be used inconjunction with other groups, such as carbamate, as in “mono- ordi-hydrocarbylcarbamate.” Mono- or di-hydrocarbylcarbamate includegroups of the formula (alkyl₁)NH(C═O)O— and (alkyl₁)N(alkyl₂)(C═O)O— aswell as groups in which one or both of the alkyl groups are replaced byan hydrocarbon group containing unsaturated carbon-carbon bonds.

A “metallocene” is a compound consisting of two 5- or 6-memberedaromatic carbocyclic groups bound to a metal center, where the metal isneutral or cationic. An example of metallocene includes, but is notlimited to, ferrocene.

The term “mono- and/or di-alkylamino” indicates secondary or tertiaryalkyl amino groups, wherein the alkyl groups are independently chosenalkyl groups, as defined herein, having the indicated number of carbonatoms. The point of attachment of the alkylamino group is on thenitrogen. Examples of mono- and di-alkylamino groups include ethylamino,dimethylamino, and methyl-propyl-amino.

“Mono- and/or di-alkylcarbamate” includes mono-alkylcarbamate groups offormula (alkyl₁)O(C═O)NH— or a dialkylcarboxamide groups of the formula(alkyl₁)O(C═O)N(alkyl₂)- in which the point of attachment of the mono-or di-alkylcarboxamide substituent to the molecule it substitutes is onthe nitrogen of the carbamate amino. The term “mono and/ordi-alkylcarbamate” also includes groups of the formula (alkyl₁)NH(C═O)O—and (alkyl₁)N(alkyl₂)(C═O)O— in which the carbamate is covalently boundto the group it substitutes by its non-keto oxygen atom. The groupsalkyl₁ and alkyl₂ are independently chosen alkyl groups, carrying thealkyl definition set forth in this disclosure and having the indicatednumber of carbon atoms.

“Mono- and/or di-alkylcarboxamide” indicates a mono-alkylcarboxamidegroup of formula (alkyls)-NH—(C═O)— or a dialkylcarboxamide group of theformula (alkyl₁)(alkyl₂)-N—(C═O)— in which the point of attachment ofthe mono- or dialkylcarboxamide substituent to the molecule itsubstitutes is on the carbon of the carbonyl group. The term “monoand/or di-alkylcarboxamide”also includes groups of the formula(alkyl₁)(C═O)NH— and (alkyl₁)(C═O)(alkyl₂)N— in which the point ofattachment is the nitrogen atom. The groups alkyl₁ and alkyl₂ areindependently chosen alkyl groups having the indicated number of carbonatoms. Likewise “mono- and/or di-alkyl sulfonamide” is any of amono-alkylsulfonamide group of formula (alkyl₁)-NH—(SO₂)—, a mono-alkylsulfonamide group of formula (alkyl₁)-(SO₂)—NH—, a dialkylsulfonamidegroup of the formula (alkyl₁)(alkyl₂)-N—(SO₂)—, and a group of theformula (alkyl₁)-(SO₂)-(alkyl₂)N—.

“Thioalkyl” is an alkyl group as defined above with the indicated numberof carbon atoms covalent bound to the group it substitutes by an sulfurbridge (—S—).

The term “substituted”, as used herein, means that any one or morehydrogens on the designated atom or group is replaced with a selectionfrom the indicated group, provided that the designated atom's normalvalence is not exceeded. When the substituent is oxo (i.e., ═O) then 2hydrogens on the atom are replaced. When an oxo group substitutesaromatic moieties, the corresponding partially unsaturated ring replacesthe aromatic ring. For example a pyridyl group substituted by oxo is apyridone. Combinations of substituents and/or variables are permissibleonly if such combinations result in stable compounds or useful syntheticintermediates. A stable compound or stable structure is meant to imply acompound that is sufficiently robust to survive isolation from areaction mixture, and subsequent formulation into an effectivetherapeutic agent. Unless otherwise specified substituents are namedinto the core structure. For example, it is to be understood that whenaminoalkyl is listed as a possible substituent the point of attachmentof this substituent to the core structure is in the alkyl portion.

Suitable groups that may be present on a “substituted” position include,but are not limited to, e.g., halogen; cyano; hydroxyl; nitro; azido;alkanoyl (such as a C₂-C₆ alkanoyl group); carboxamide; alkyl groups(including cycloalkyl groups) having 1 to about 8 carbon atoms, or 1 toabout 6 carbon atoms; alkenyl and alkynyl groups including groups havingone or more unsaturated linkages and from 2 to about 8, or 2 to about 6carbon atoms; alkoxy groups having one or more oxygen linkages and from1 to about 8, or from 1 to about 6 carbon atoms; aryloxy such asphenoxy; alkylthio groups including those having one or more thioetherlinkages and from 1 to about 8 carbon atoms, or from 1 to about 6 carbonatoms; alkylsulfinyl groups including those having one or more sulfinyllinkages and from 1 to about 8 carbon atoms, or from 1 to about 6 carbonatoms; alkylsulfonyl groups including those having one or more sulfonyllinkages and from 1 to about 8 carbon atoms, or from 1 to about 6 carbonatoms; aminoalkyl groups including groups having one or more N atoms andfrom 1 to about 8, or from 1 to about 6 carbon atoms; aryl having 6 ormore carbons and one or more rings, (e.g., phenyl, biphenyl, naphthyl,or the like, each ring either substituted or unsubstituted aromatic);arylalkyl having 1 to 3 separate or fused rings and from 6 to about 18ring carbon atoms, with benzyl being an exemplary arylalkyl group;arylalkoxy having 1 to 3 separate or fused rings and from 6 to about 18ring carbon atoms, with benzyloxy being an exemplary arylalkoxy group;or a saturated, unsaturated, or aromatic heterocyclic group having 1 to3 separate or fused rings with 3 to about 8 members per ring and one ormore N, O or S atoms, e.g. coumarinyl, quinolinyl, isoquinolinyl,quinazolinyl, pyridyl, pyrazinyl, pyrimidinyl, furanyl, pyrrolyl,thienyl, thiazolyl, triazinyl, oxazolyl, isoxazolyl, imidazolyl,indolyl, benzofuranyl, benzothiazolyl, tetrahydrofuranyl,tetrahydropyranyl, piperidinyl, morpholinyl, piperazinyl, andpyrrolidinyl. Such heterocyclic groups may be further substituted, e.g.with hydroxy, alkyl, alkoxy, halogen and amino.

A “dosage form” means a unit of administration of an active agent.Examples of dosage forms include tablets, capsules, injections,suspensions, liquids, emulsions, creams, ointments, suppositories,inhalable forms, transdermal forms, and the like.

“Pharmaceutical compositions” are compositions comprising at least oneactive agent, such as a compound or salt of Formula I, and at least oneother substance, such as a carrier. Pharmaceutical compositions optionalcontain one or more additional active agents. When specified,pharmaceutical compositions meet the U.S. FDA's GMP (good manufacturingpractice) standards for human or non-human drugs. “Pharmaceuticalcombinations” are combinations of at least two active agents which maybe combined in a single dosage form or provided together in separatedosage forms with instructions that the active agents are to be usedtogether to treat a disorder, such as hepatitis C.

“Pharmaceutically acceptable salts” includes derivatives of thedisclosed compounds in which the parent compound is modified by makinginorganic and organic, non-toxic, acid or base addition salts thereof.The salts of the present compounds can be synthesized from a parentcompound that contains a basic or acidic moiety by conventional chemicalmethods. Generally, such salts can be prepared by reacting free acidforms of these compounds with a stoichiometric amount of the appropriatebase (such as Na, Ca, Mg, or K hydroxide, carbonate, bicarbonate, or thelike), or by reacting free base forms of these compounds with astoichiometric amount of the appropriate acid. Such reactions aretypically carried out in water or in an organic solvent, or in a mixtureof the two. Generally, non-aqueous media like ether, ethyl acetate,ethanol, isopropanol, or acetonitrile are preferred, where practicable.Salts of the present compounds further include solvates of the compoundsand of the compound salts.

Examples of pharmaceutically acceptable salts include, but are notlimited to, mineral or organic acid salts of basic residues such asamines; alkali or organic salts of acidic residues such as carboxylicacids; and the like. The pharmaceutically acceptable salts include theconventional non-toxic salts and the quaternary ammonium salts of theparent compound formed, for example, from non-toxic inorganic or organicacids. For example, conventional non-toxic acid salts include thosederived from inorganic acids such as hydrochloric, hydrobromic,sulfuric, sulfamic, phosphoric, nitric and the like; and the saltsprepared from organic acids such as acetic, propionic, succinic,glycolic, stearic, lactic, malic, tartaric, citric, ascorbic, pamoic,maleic, hydroxymaleic, phenylacetic, glutamic, benzoic, salicylic,mesylic, esylic, besylic, sulfanilic, 2-acetoxybenzoic, fumaric,toluenesulfonic, methanesulfonic, ethane disulfonic, oxalic, isethionic,HOOC—(CH₂)_(n)—COOH where n is 0-4, and the like. Lists of additionalsuitable salts may be found, e.g., in Remington's PharmaceuticalSciences, 17th ed., Mack Publishing Company, Easton, Pa., p. 1418(1985).

The term “carrier” applied to pharmaceutical compositions/combinationsof the invention refers to a diluent, excipient, or vehicle with whichan active compound is provided.

A “pharmaceutically acceptable excipient” means an excipient that isuseful in preparing a pharmaceutical composition/combination that isgenerally safe, non-toxic and neither biologically nor otherwiseundesirable, and includes an excipient that is acceptable for veterinaryuse as well as human pharmaceutical use. A “pharmaceutically acceptableexcipient” as used in the present application includes both one and morethan one such excipient.

A “patient” is a human or non-human animal in need of medical treatment.Medical treatment can include treatment of an existing condition, suchas a disease or disorder, prophylactic or preventative treatment, ordiagnostic treatment. In some embodiments the patient is a humanpatient.

“Providing” means giving, administering, selling, distributing,transferring (for profit or not), manufacturing, compounding, ordispensing.

“Providing a compound of Formula I with at least one additional activeagent” means the compound of Formula I and the additional activeagent(s) are provided simultaneously in a single dosage form, providedconcomitantly in separate dosage forms, or provided in separate dosageforms for administration separated by some amount of time that is withinthe time in which both the compound of Formula I and the at least oneadditional active agent are within the blood stream of a patient. Incertain embodiments the compound of Formula I and the additional activeagent need not be prescribed for a patient by the same medical careworker. In certain embodiments the additional active agent or agentsneed not require a prescription. Administration of the compound ofFormula I or the at least one additional active agent can occur via anyappropriate route, for example, oral tablets, oral capsules, oralliquids, inhalation, injection, suppositories or topical contact.

“Treatment,” as used herein includes providing a compound of Formula I,either as the only active agent or together with at least one additionalactive agent sufficient to: (a) prevent a disease or a symptom of adisease from occurring in a patient who may be predisposed to thedisease but has not yet been diagnosed as having it (e.g. includingdiseases that may be associated with or caused by a primary disease (asin liver fibrosis that can result in the context of chronic HCVinfection); (b) inhibiting the disease, i.e. arresting its development;and (c) relieving the disease, i.e., causing regression of the disease.“Treating” and “treatment” also means providing a therapeuticallyeffective amount of a compound of Formula I, as the only active agent ortogether with at least one additional active agent to a patient havingor susceptible to a hepatitis C infection.

A “therapeutically effective amount” of a pharmaceuticalcomposition/combination of this invention means an amount effective,when administered to a patient, to provide a therapeutic benefit such asan amelioration of symptoms, e.g., an amount effective to decrease thesymptoms of a hepatitis C infection. For example a patient infected witha hepatitis C virus may present elevated levels of certain liverenzymes, including AST and ALT. Normal levels of AST are from 5 to 40units per liter of serum (the liquid part of the blood) and normallevels of ALT are from 7 to 56 units per liter of serum. Atherapeutically effect amount is thus an amount sufficient to provide asignificant reduction in elevated AST and ALT levels or an amountsufficient to provide a return of AST and ALT levels to the normalrange. A therapeutically effective amount is also an amount sufficientto prevent a significant increase or significantly reduce the detectablelevel of virus or viral antibodies in the patient's blood, serum, ortissues. One method of determining treatment efficacy includes measuringHCV RNA levels by a conventional method for determining viral RNA levelssuch as the Roche TaqMan assay. In certain preferred embodimentstreatment reduces HCV RNA levels below the limit of quantitation (30IU/mL, as measured by the Roche TaqMan® assay) or more preferably belowthe limit of detection (10 IU/mL, Roche TaqMan).

A significant increase or reduction in the detectable level of virus orviral antibodies is any detectable change that is statisticallysignificant in a standard parametric test of statistical significancesuch as Student's T-test, where p<0.05.

Chemical Description

Formula I and Formula II (shown below) include all subformulae thereof.In certain situations, the compounds of Formula I or Formula II maycontain one or more asymmetric elements such as stereogenic centers,stereogenic axes and the like, e.g. asymmetric carbon atoms, so that thecompounds can exist in different stereoisomeric forms. These compoundscan be, for example, racemates or optically active forms. For compoundswith two or more asymmetric elements, these compounds can additionallybe mixtures of diastereomers. For compounds having asymmetric centers,it should be understood that all of the optical isomers and mixturesthereof are encompassed. In addition, compounds with carbon-carbondouble bonds may occur in Z- and E-forms, with all isomeric forms of thecompounds being included in the present invention. In these situations,single enantiomers, i.e., optically active forms, can be obtained byasymmetric synthesis, synthesis from optically pure precursors, or byresolution of the racemates. Resolution of the racemates can also beaccomplished, for example, by conventional methods such ascrystallization in the presence of a resolving agent, or chromatography,using, for example using a chiral HPLC column.

Where a compound exists in various tautomeric forms, the invention isnot limited to any one of the specific tautomers, but rather includesall tautomeric forms.

The present invention is intended to include all isotopes of atomsoccurring in the present compounds. Isotopes include those atoms havingthe same atomic number but different mass numbers. By way of generalexample, and without limitation, isotopes of hydrogen include tritiumand deuterium and isotopes of carbon include ¹¹C, ¹³C, and ¹⁴C.

Certain compounds are described herein using a general formula thatincludes variables, e.g. D, M, A, P, J, and W. Unless otherwisespecified, each variable within such a formula is defined independentlyof other variables. Thus, if a group is said to be substituted, e.g.with 0-2 R*, then the group may be substituted with up to two R* groupsand R* at each occurrence is selected independently from the definitionof R*. Also, combinations of substituents and/or variables arepermissible only if such combinations result in stable compounds.

In addition the compounds and salts of Formula I discussed in theSUMMARY section, the disclosure includes compounds and salt of FormulaI, and pharmaceutical compositions/combinations of such compounds inwhich the variables meet any of the following conditions.

(i) D-M-D is any ofT-R-J¹-A-W-J¹-R-T;T-R-J¹-A-J¹-R-T;T-R-J²-A-J²-R-T;T-R-J¹-W-A-J²-R-T; andT-R-J¹-A-J²-R-T.

(ii) A is

(iii) A is

(iv) A is

(v) A is

(vi) A is

(vii) A is

(viii) A is

(ix) A is

(x) A is

(xi) A is

(xii) A is

(xiii) A is

(xiv) A is

(xv) At least one P is J¹-W; and W is phenyl, optionally substitutedwith one or more substituents independently chosen from halogen,C₁-C₂alkyl, and C₁-C₂alkoxy.

(xvi) P is J-W or J, wherein at least one J is J¹ and J¹ is

(xvii) At least one P is J¹ and J¹ is

(xviii) At least one P is J² and J² is a benzimidazole group, optionallysubstituted with one or more substituents independently chosen fromhalogen, C₁-C₂alkyl, and C₁-C₂alkoxy.

(xix) R is independently chosen from

each of which is optionally substituted with one or more substituentsindependently chosen from halogen, C₁-C₄alkyl, and C₁-C₄alkoxy.

(xx) R is independently chosen from

(xxi) T is independently chosen C₂-C₆alkanoyl substituted with mono- anddi-C₁-C₆alkylcarbamate, each of which T is optionally substituted with(C₁-C₄thioalkyl)C₀-C₄alkyl.

(xxii) Also included are compounds and salts of the formulaT-R-J²-A-J²-R-T.

A, in the formula T-R-J²-A-J²-R-T, is a group of the formula

J² is an 8- to 10-membered heteroaryl group containing for 2 heteroatomsindependently chosen from N, O, and S, wherein J² is optionallysubstituted with one or more substituents independently chosen fromamino, cyano, hydroxyl, halogen, C₁-C₄alkyl, C₁-C₄alkoxy, mono- anddi-C₁-C₄alkylamino, C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

Each R is an independently chosen 8- to 10-membered bicyclic ringsystems containing one or two nitrogen atoms with remaining ring atomsbeing carbon, which 8- to 10-membered bicyclic ring is saturated orcontains 1 unsaturated bond; and R is optionally substituted with one ormore substituents independently chosen from cyano, hydroxyl, halogen,C₁-C₂alkyl, C₁-C₂alkoxy, C₁-C₂haloalkyl, C₁-C₂haloalkyl,C₁-C₂haloalkylene, and C₁-C₂alkylsulfonyl.

T² is independently chosen at each occurrence from C₂-C₆alkanoyl,C₁-C₆alkylester, C₁-C₆alkenylester, C₁-C₆alkylsulfonamide,C₁-C₆alkylsulfonyl, C₂-C₆alkanoyl substituted with mono- ordi-C₁-C₆hydrocarbylcarbamate, C₂-C₆alkanoyl substituted with urea ormono- or di-C₁-C₆alkylurea, and C₂-C₆alkanoyl substituted with mono- ordi-C₁-C₆alkylcarboxamide, each of which T² is optionally substitutedwith 1 or more substituents independently chosen from amino, cyano,hydroxyl, halogen, (C₁-C₄alkoxy)C₀-C₄alkyl, (mono- anddi-C₁-C₄alkylamino)C₀-C₄alkyl, C₁-C₆alkyl, (C₁-C₄thioalkyl)C₀-C₄alkyl,C₃-C₇cycloalkyl, phenyl, C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

In certain embodiments of xxii, it is preferred that -J²-R— is a groupof the formula

or more particularly

J²-R— may be unsubstituted or substituted with one or more substituentsindependently chosen from amino, cyano, hydroxyl, halogen, C₁-C₄alkyl,C₁-C₄alkoxy, mono- and di-C₁-C₄alkylamino, C₁-C₂haloalkyl, andC₁-C₂haloalkoxy.

In certain embodiments of xxii, it is also preferred that T² isC₂-C₆alkanoyl substituted with mono- or di-C₁-C₆hydrocarbylcarbamate,which T² is optionally substituted with 1 or more substituentsindependently chosen from amino, cyano, hydroxyl, halogen,(C₁-C₄alkoxy)C₀-C₄alkyl, (mono- and di-C₁-C₄alkylamino)C₀-C₄alkyl,C₁-C₆alkyl, (C₁-C₄thioalkyl)C₀-C₄alkyl, C₃-C₇cycloalkyl, phenyl,C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

Any of the preceding conditions for compounds of Formula I may be usedtogether to define a subgeneric formula of Formula I so long as a stablecompound results. All such subgeneric formulas are included in thisdisclosure.

In addition the compounds and salts of Formula I discussed in theSUMMARY section, the disclosure includes compositions and combinationsof Formula I and Formula II, wherein Formula II is:

Within Formula II the variables R₁′, R₂′, R₃′, R₄′, R₆′, R₇′, R₈′, R₁₆,and T′ carry the definitions set forth below.

R₁′ and R₂′ are joined to form a 5- to 7-membered heterocycloalkyl ringcontaining 1 or 2 heteroatoms independently chosen from N, O, and Swhich ring is optionally fused to a phenyl or 5- or 6-memberedheteroaryl to form a bicyclic ring system, each of which 5- to7-membered heterocycloalkyl ring or bicyclic ring system is optionallysubstituted.

For the variables R₃′-R₈′ one of the following conditions is met. R₃′,R₄′, R₅′, and R₆′ are independently hydrogen, C₁-C₄alkyl, or(C₃-C₇cycloalkyl)C₀-C₄alkyl; and R₇′ and R₈′ are joined to form anoptionally substituted 3- to 7-membered cycloalkyl ring.

R₃′, R₄′, and R₆′ are independently hydrogen, C₁-C₄alkyl, or(C₃-C₇cycloalkyl)C₀-C₄alkyl; and R₈′ is hydrogen or C₁-C₄alkyl; and R₅′is joined to R₇′ by a C₆-C₁₀ saturated or unsaturated hydrocarbon chain.

R₃′, R₄′, and R₆′ are independently hydrogen, C₁-C₄alkyl, or(C₃-C₇cycloalkyl)C₀-C₄alkyl; and R₇′ and R₈′ are joined to form anoptionally substituted 3- to 7-membered cycloalkyl ring; and R₅′ isjoined to the 3- to 7-membered optionally substituted cycloalkyl ringformed by R₇ and R₈ by a C₆-C₁₀ saturated, partially unsaturated orunsaturated hydrocarbon chain.

T′ is a group of the formula:

R₉ is hydroxyl, amino, —COOH, —NR₁₀R₁₁, —OR₁₂, —NR₁₀(S═O)R₁₁, or—NR₁₀SO₂R₁₁. R₁₀, R₁₁, and R₁₂ are independently at each occurrencehydrogen, or C₁-C₆alkyl, C₂-C₆alkenyl, (aryl)C₀-C₂alkyl,(C₃-C₇cycloalkyl)C₀-C₂alkyl, (heterocycloalkyl)C₀-C₂alkyl, or (5- to10-membered heteroaryl)C₀-C₂alkyl, each of which is optionallysubstituted with 1 to 3 substituents independently chosen from halogen,hydroxyl, oxo, C₁-C₂alkyl, C₁-C₂alkoxy, trifluoromethyl, andtrifluoromethoxy:

Z′ is

where X₁, X₂, X₃, X₄, and X₅ are independently N or CH and no more thantwo of X₁-X₅ are N.

R₂₁ represents from 0 to 3 groups independently chosen from halogen,hydroxyl, amino, cyano, —CONH₂, —COOH, C₁-C₄alkyl, C₂-C₄alkanoyl,C₁-C₄alkoxy, C₁-C₄alkylthio, mono- and di-C₁-C₄alkylamino,C₁-C₂haloalkyl, and C₁-C₂haloalkoxy.

R₂₂ is hydrogen, halogen, hydroxyl, amino, cyano, —CONH₂, —COOH,C₁-C₄alkyl, C₂-C₄alkanoyl, C₁-C₄alkoxy, C₁-C₄alkylthio, mono- anddi-C₁-C₄alkylamino, C₁-C₄alkylester, C₁-C₂haloalkyl, andC₁-C₂haloalkoxy; or R₂₂ is (C₃-C₇cycloalkyl)C₀-C₂alkyl,(phenyl)C₀-C₂alkyl, (phenyl)C₀-C₂alkoxy, (5- or 6-memberedheteroaryl)C₀-C₂alkyl, (5- or 6-membered heteroaryl)C₀-C₂alkoxy,naphthyl, indanyl, (5- or 6-membered heterocycloalkyl)C₀-C₂alkyl, or 9-or 10 membered bicyclic heteroaryl, each of which is substituted with 0,1, or 2 substituents independently chosen from

(i) halogen, hydroxyl, amino, cyano, nitro, —COOH, —CONH₂, CH₃(C═O)NH—,C₁-C₄alkyl, C₁-C₄alkoxy, C₁-C₄hydroxyalkyl, mono- anddi-C₁-C₄alkylamino, —NR₈SO₂R₁₁, —C(O)OR₁₁, —NR₈COR₁₁, —NR₈C(O)OR₁₁,trifluoromethyl, trifluoromethoxy, and

(ii) phenyl and 5- or 6-membered heteroaryl, each of which issubstituted with 0 or 1 or more of halogen, hydroxyl, C₁-C₄alkyl, andC₁-C₂alkoxy; wherein R₈ is hydrogen, C₁-C₄alkyl, or C₃C₆cycloalkyl, andR₁₁ is as defined above.

R₁₆ represents 0 to 4 substituents is independently chosen at fromhalogen, C₁-C₂alkyl, and C₁-C₂alkoxy.

Any of the preceding conditions for compounds of Formula I may be usedtogether to define a subgeneric formula of Formula I so long as a stablecompound results and all such subgeneric formulas are included in thisdisclosure.

This disclosure also includes pharmaceutical compositions andcombinations comprising a compound of Formula I and a compound ofFormula II. As well as methods of treatment comprising administeringsuch compositions/combinations to a patient infected with hepatitis C.

For example the disclosure includes compositions and combinations inwhich the compound of Formula II is

This disclosure also includes pharmaceutical compositions andcombinations comprising a compound of Formula I and a compound ofFormula II. As well as methods of treatment comprising administeringsuch compositions/combinations to a patient infected with hepatitis C.

For example the disclosure includes compositions and combinations inwhich the compound of Formula II is

NS3a protease inhibitors of Formula II, useful in the pharmaceuticalcompositions and combinations described here have been disclosedpreviously. U.S. Pat. No. 7,906,619, issued Mar. 15, 2011, is herebyincorporated by reference in its entirety for its teachings regarding4-amino-4-oxobutanoyl peptides. The '619 patent is particularlyincorporated by reference at the Examples section beginning in column 50and extending to column 85 which discloses compounds useful incompositions/combination with Compounds of Formula I described here.

Published US Pat. Appl. No. 2010-0216725, published Aug. 26, 2010, ishereby incorporated by reference in its entirety for its teachingsregarding 4-amino-4-oxobutanoyl peptides. The '725 application isparticularly incorporated by reference at the Examples section beginningat page 22 and extending to page 100 which discloses compounds useful incompositions/combination with Compounds of Formula I described here.

Published US Pat. Appl. No. 2010-0152103, published Jun. 17, 2010, ishereby incorporated by reference in its entirety for its teachingsregarding 4-amino-4-oxobutanoyl peptide cyclic analogues. The '103application is particularly incorporated by reference at the Examplessection beginning at page 19 and extending to page 60 which disclosescompounds useful in compositions/combination with Compounds of Formula Idescribed here.

Pharmaceutical Preparations

Compounds disclosed herein can be administered as the neat chemical, butare preferably administered as a pharmaceutical composition.Accordingly, the disclosure provides pharmaceutical compositionscomprising a compound or pharmaceutically acceptable salt of Formula I,together with at least one pharmaceutically acceptable carrier. Thepharmaceutical composition/combination may contain a compound or salt ofFormula I as the only active agent, but is preferably contains at leastone additional active agent. In certain embodiments it is preferred thatthe additional active agent is an NS3a protease inhibitor, such as acompound of salt of Formula II. In certain embodiments thepharmaceutical composition is in a dosage form that contains from about0.1 mg to about 2000 mg, from about 10 mg to about 1000 mg, from about100 mg to about 800 mg, or from about 200 mg to about 600 mg of acompound of Formula I and optionally from about 0.1 mg to about 2000 mg,from about 10 mg to about 1000 mg, from about 100 mg to about 800 mg, orfrom about 200 mg to about 600 mg of an additional active agent in aunit dosage form. The pharmaceutical composition may also include amolar ratio of a compound of Formula I and an additional active agent.For example the pharmaceutical composition may contain a molar ratio ofabout 0.5:1, about 1:1, about 2:1, about 3:1 or from about 1.5:1 toabout 4:1 of an NS3a protease inhibitor of Formula II to NS5a inhibitorof Formula I.

Compounds disclosed herein may be administered orally, topically,parenterally, by inhalation or spray, sublingually, transdermally, viabuccal administration, rectally, as an ophthalmic solution, or by othermeans, in dosage unit formulations containing conventionalpharmaceutically acceptable carriers. The pharmaceutical composition maybe formulated as any pharmaceutically useful form, e.g., as an aerosol,a cream, a gel, a pill, a capsule, a tablet, a syrup, a transdermalpatch, or an ophthalmic solution. Some dosage forms, such as tablets andcapsules, are subdivided into suitably sized unit doses containingappropriate quantities of the active components, e.g., an effectiveamount to achieve the desired purpose.

Carriers include excipients and diluents and must be of sufficientlyhigh purity and sufficiently low toxicity to render them suitable foradministration to the patient being treated. The carrier can be inert orit can possess pharmaceutical benefits of its own. The amount of carrieremployed in conjunction with the compound is sufficient to provide apractical quantity of material for administration per unit dose of thecompound.

Classes of carriers include, but are not limited to binders, bufferingagents, coloring agents, diluents, disintegrants, emulsifiers,flavorants, glidents, lubricants, preservatives, stabilizers,surfactants, tableting agents, and wetting agents. Some carriers may belisted in more than one class, for example vegetable oil may be used asa lubricant in some formulations and a diluent in others. Exemplarypharmaceutically acceptable carriers include sugars, starches,celluloses, powdered tragacanth, malt, gelatin; talc, and vegetableoils. Optional active agents may be included in a pharmaceuticalcomposition, which do not substantially interfere with the activity ofthe compound of the present invention.

The pharmaceutical compositions/combinations can be formulated for oraladministration. These compositions contain between 0.1 and 99 weight %(wt. %) of a compound of Formula I and usually at least about 5 wt. % ofa compound of Formula. Some embodiments contain from about 25 wt. % toabout 50 wt. % or from about 5 wt. % to about 75 wt. % of the compoundof Formula.

Methods of Treatment

The pharmaceutical compositions/combinations disclosed herein are usefulfor treating hepatitis C infections in patients.

This disclosure provides methods of treating viral infections, includinghepatitis C infections, by providing an effective amount of a compoundor pharmaceutically acceptable salt of Formula I to patient infectedwith a hepatitis C virus. A compound or salt of Formula I may beprovided as the only active agent or may be provided together with oneor more additional active agents. In certain embodiments the compound orsalt of Formula I is administered together with a compound or salt ofFormula II or other NS3a protease inhibitor. In certain embodiment thepharmaceutical composition contains a compound of Formula I togetherwith an NS5b inhibitor, and optionally an additional active agent.

An effective amount of a pharmaceutical composition/combination of theinvention may be an amount sufficient to (a) inhibit the progression ofhepatitis C; (b) cause a regression of the hepatitis C infection; or (c)cause a cure of a hepatitis C infection such that HCV virus or HCVantibodies can no longer be detected in a previously infected patient'sblood or plasma. An amount of a pharmaceutical composition/combinationeffective to inhibit the progress or cause a regression of hepatitis Cincludes an amount effective to stop the worsening of symptoms ofhepatitis C or reduce the symptoms experienced by a patient infectedwith the hepatitis C virus. Alternatively a halt in progression orregression of hepatitis C may be indicated by any of several markers forthe disease. For example, a lack of increase or reduction in thehepatitis C viral load or a lack of increase or reduction in the numberof circulating HCV antibodies in a patient's blood are markers of a haltin progression or regression of hepatitis C infection. Other hepatitis Cdisease markers include aminotransferase levels, particularly levels ofthe liver enzymes AST and ALT. Normal levels of AST are from 5 to 40units per liter of serum (the liquid part of the blood) and normallevels of ALT are from 7 to 56 units per liter of serum. These levelswill typically be elevated in a HCV infected patient. Disease regressionis usually marked by the return of AST and ALT levels to the normalrange.

Symptoms of hepatitis C that may be affected by an effective amount of apharmaceutical composition/combination of the invention includedecreased liver function, fatigue, flu-like symptoms: fever, chills,muscle aches, joint pain, and headaches, nausea, aversion to certainfoods, unexplained weight loss, psychological disorders includingdepression, tenderness in the abdomen, and jaundice.

“Liver function” refers to a normal function of the liver, including,but not limited to, a synthetic function including synthesis of proteinssuch as serum proteins (e.g., albumin, clotting factors, alkalinephosphatase, aminotransferases (e.g., alanine transaminase, aspartatetransaminase), 5′-nucleosidase, y glutaminyltranspeptidase, etc.),synthesis of bilirubin, synthesis of cholesterol, and synthesis of bileacids; a liver metabolic function, including carbohydrate metabolism,amino acid and ammonia metabolism, hormone metabolism, and lipidmetabolism; detoxification of exogenous drugs; and a hemodynamicfunction, including splanchnic and portal hemodynamics.

An effective amount of a pharmaceutical composition/combinationdescribed herein will also provide a sufficient concentration of theactive agents in the concentration when administered to a patient. Asufficient concentration of an active agent is a concentration of theagent in the patient's body necessary to prevent or combat theinfection. Such an amount may be ascertained experimentally, for exampleby assaying blood concentration of the agent, or theoretically, bycalculating bioavailability. The amount of an active agent sufficient toinhibit viral infection in vitro may be determined with a conventionalassay for viral infectivity such as a replicon based assay, which hasbeen described in the literature.

Pharmaceutical compositions/combinations and methods of treatment inwhich a compound or salt of Formula I is provided together with one ormore additional active agents are included herein. In preferredembodiments a compound of Formula I is provided together with an NS3aprotease inhibitor, either in a single pharmaceutical composition or ain separate dosage forms with instructions to the patient to use thecompound of Formula I and additional active agent together. Compounds ofFormula II and compounds disclosed in U.S. Pat. No. 7,906,619, US Pat.Appl. No. 2010-0216725, and US Pat. Appl. No. 2010-0152103, most ofwhich are within the scope of Formula II, are suitable NS3a proteaseinhibitors for use in combination with compounds and salts of Formula I.In certain embodiments the active agent (or agents) is an HCV proteaseinhibitor or HCV polymerase inhibitor. For example the proteaseinhibitor may be telaprevir (VX-950) and the polymerase inhibitor may bevalopicitabine, or NM 107, the active agent which valopicitabine isconverted into in vivo. In certain embodiments the at least oneadditional active agent is ribavirin, interferon, or Peg-interferonalpha conjugate. In certain embodiments the at least one additionalactive agent is ACH-1625 or ACH-2684.

According to the methods of the invention, the compound orpharmaceutically acceptable salt of Formula I and at least oneadditional active agent may be: (1) co-formulated and administered ordelivered simultaneously in a combined formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by any othercombination therapy regimen known in the art. When delivered inalternation therapy, the methods of the invention may compriseadministering or delivering the compound or salt of Formula I and anadditional active agent sequentially, e.g., in separate solution,emulsion, suspension, tablets, pills or capsules, or by differentinjections in separate syringes. In general, during alternation therapy,an effective dosage of each active ingredient is administeredsequentially, i.e., serially, whereas in simultaneous therapy, effectivedosages of two or more active ingredients are administered together.Various sequences of intermittent combination therapy may also be used.

Methods of treatment and pharmaceutical combinations including compoundsor pharmaceutically acceptable salts of Formula I described hereintogether with any one or combination of the following compounds andsubstances as an additional active agent are provided by the disclosure:

Anti-fibrotics: IP-501 (InterMune)

Caspase Inhibitors: IDN-6556 (Idun Pharmaceuticals) and GS-9450 (Gilead)

Cyclophilin Inhibitors: for example, NIM811 (Novartis), SCY-635(Scynexis), and DEBIO-025 (Debiopharm);

Cytochrome P450 monooxygenase inhibitors: ritonavir, ketoconazole,troleandomycin, 4-methyl pyrazole, cyclosporin, clomethiazole,cimetidine, itraconazole, fluconazole, miconazole, fluvoxamine,fluoxetine, nefazodone, sertraline, indinavir, nelfinavir, amprenavir,fosamprenavir, saquinavir, lopinavir, delavirdine, erythromycin, VX-944,and VX-497 (Merimebodib). Preferred CYP inhibitors include ritonavir,ketoconazole, troleandomycin, 4-methyl pyrazole, cyclosporin, andclomethiazole;

Glucocorticoids: hydrocortisone, cortisone, prednisone, prednisolone,methylprednisolone, triamcinolone, paramethasone, betamethasone, anddexamethasone.

HCV Protease Inhibitors: for example ACH-1625 and ACH-2684. U.S. Pat.No. 7,906,619 is hereby incorporated for its teachings regardinganti-HCV compounds. U.S. patent application Ser. No. 12/635,270 at pages52-167 and Ser. No. 12/635,049 at pages 43-92 are hereby incorporated byreference for their teachings regarding anti-HCV compounds, ACH 1625 andACH 2684 (Achillion), ABT-450 (Abbott), ACL-181 and AVL-192 (Avila),BI-335 (Boehringer Ingelheim), BMS-032 (Bristol Meyers Squibb),boceprevir (Merck), TMC-435, MK-7152 (Merck), GS-9256 (Gilead), GS-9451(Gilead), R7227 (Intermune), VX-500 (Vertex), VX-950 (telaprevir,Vertex), VX-985 (Vertex), TMC-435 (Tibotec), GW-433908 (prodrug ofAmprenavir, Glaxo/Vertex), indinavir (CRIXIVAN, Merck), ITMN-191(Intermune/Array Biopharma), BILN 2061 (Boehringer-Ingelheim), TMC435350(Tibotec/Medivir), BI 201335 (Boehringer Ingelheim), PHX-1766(Phenomix), MK-7009 (Merck), narlaprevir (SCH900518, Schering)

Hematopoietins: hematopoietin-1 and hematopoietin-2. Other members ofthe hematopoietin superfamily such as the various colony stimulatingfactors (e.g. G-CSF, GM-CSF, M-CSF), Epo, and SCF (stem cell factor)

Homeopathic Therapies: Milk Thistle, silymarin, ginseng, glycyrrhizin,licorice root, schisandra, vitamin C, vitamin E, beta carotene, andselenium

Immunomodulatory compounds: thalidomide, IL-2, hematopoietins, IMPDHinhibitors, for example Merimepodib (Vertex Pharmaceuticals Inc.),interferon, including natural interferon (such as OMNIFERON, Viragen andSUMIFERON, Sumitomo, a blend of natural interferons), natural interferonalpha (ALFERON, Hemispherx Biopharma, Inc.), interferon alpha nl fromlymphblastoid cells (WELLFERON, Glaxo Wellcome), oral alpha interferon,Peg-interferon, Peg-interferon alfa 2a (PEGASYS, Roche), recombinantinterferon alfa 2a (ROFERON, Roche), inhaled interferon alpha 2b (AERX,Aradigm), Peg-interferon alpha 2b (ALBUFERON, Human GenomeSciences/Novartis, PEGINTRON, Schering), recombinant interferon alfa 2b(INTRON A, Schering), pegylated interferon alfa 2b (PEG-INTRON,Schering, VIRAFERONPEG, Schering), interferon beta-1a (REBIF,Ares-Serono, Inc. and Pfizer), consensus interferon alpha (INFERGEN,Intermune), interferon gamma-1b (ACTIMMUNE, Intermune, Inc.),un-pegylated interferon alpha, alpha interferon, and its analogs, andsynthetic thymosin alpha 1 (ZADAXIN, SciClone Pharmaceuticals Inc.), andlamdba interferon (BMS)

Immunosupressants: sirolimus (RAPAMUNE, Wyeth)

Interleukins: (IL-1, IL-3, IL-4, IL-5, IL-6, IL-10, IL-11, IL-12), LIF,TGF-beta, TNF-alpha) and other low molecular weight factors (e.g.AcSDKP, pEEDCK, thymic hormones, and minicytokines)

Interferon Enhancers: EMZ702 (Transition Therapeutics)

IRES inhibitors: VGX-410C (VGX Pharma)

Monoclonal and Polyclonal antibodies: XTL-6865 (HEPX-C, XTL), HuMax-HepC(Genmab), Hepatitis C Immune Globin (human) (CIVACIR, NabiBiopharmceuticals), XTL-002 (XTL), Rituximab (RITUXAN, Genentech/IDEC)

Nucleoside analogues: IDX-184 (Idenix), PSI-7977 and PSI-938(Pharmasset), INX-189 (Inhibitex), R7128 (Roche), R7348 (Roche), GS-6620(Gilead), TMC-649 (Tibotec), Lamivudine (EPIVIR, 3TC, GlaxoSmithKline),MK-0608 (Merck), zalcitabine (HIVID, Roche US Pharmaceuticals),ribavirin (including COPEGUS (Roche), REBETOL (Schering), VILONA (ICNPharmaceuticals, and VIRAZOLE (ICN Pharmaceuticals), isatoribine (AnadysPharmaceuticals), ANA971 (Anadys Pharmaceuticals), ANA245 (AnadysPharmaceuticals), and viramidine (ICN), an amidine prodrug of ribavirin.Combinations of nucleoside analogues may also be employed.

Non-nucleoside inhibitors: PSI-6130 (Roche/Pharmasset), ABT-333 andABT-072 (Abbott), delaviridine (RESCRIPTOR, Pfizer), PF-868554 (Pfizer),GSK-852 (GlaxoSmithKline), IDX-325 (Idenix), ANA-598 (Anadys), VX-222and VX-759 (Vertex), MK-3281 (Merck), BI-127 (Boehringer Ingelheim),BMS-325 (Bristol Meyers), and HCV-796 (Viropharm)

NS4a inhibitors: for example ACH-1095. US patent application no.US2007/0004711 is hereby incorporated by reference in its entirety forits teachings regarding HCV inhibitors and U.S. patent application Ser.No. 12/125,554 at pages 45-90 is hereby incorporated by reference forits teachings regarding HCV inhibitors.

NS4b inhibitors: clemizole (Arrow Therapeutics)

NS5a inhibitors: A-382 (Arrow Therapeutics), BMS-790052 (BMS) NS5binhibitors: INX-181, IDX-375, MK-3281, PSI-7977, PSI-7851, PSI-938,RG-9190, VX-222 (Vertex), and BMS-791325 (Bristol Meyers Squibb).

P7 protein inhibitor: amantadine (SYMMETREL, Endo Pharmaceuticals, Inc.)

Polymerase inhibitors: Filibuvir (PF-00868554, Pfizer), NM283(valopicitabine) (Idenix), JTK 003 (AKROS Pharma), HCV-796(ViroPharma/Wyeth), IDX184 (Idenix), VCH-916 (Vertx), R7128 (PSI6130,Roche), R1626 (Roche), MK-3281 (Merck), PSI-7851 (Pharmasset), ANA598(Anadys), BI207127 (Boehringer-Ingelheim), GS 9190 (Gilead).

RNA interference: SIRNA-034 RNAi (Sirna Therapeutics) and ISI 14803(Isis Pharmaceutical/Elan)

Therapeutic Vaccines: IC₄₁ (Intercell), IMN-0101 (Imnogenetics), GI 5005(Globeimmune), Chronvac-C (Tripep/Inovio), ED-002 (Imnogenetics),Hepavaxx C (ViRex Medical)

TNF agonists: adalimumab (HUMIRA, Abbott), entanercept (ENBREL, Amgenand Wyeth), infliximab (REMICADE, Centocor, Inc.)

Tubulin inhibitors: Colchicine

Sphingosine-1-phosphate receptor modulators: FTY720 (Novartis)

TLR agonists: ANA-975 (Anadys Pharmaceuticals), TLR7 agonist (AnadysPharmaceuticals), CPG10101 (Coley), and TLR9 agonists including CPG 7909(Coley).

Vaccines: HCV/MF59 (Chiron), IC₄₁ (Intercell), E-1 (Innogenetics)

Patients receiving hepatitis C medications are typically giveninterferon together with another active agent. Thus methods of treatmentand pharmaceutical combinations in which a compound of The invention isprovided together with an interferon, such as pegylated interferon alfa2a, as the additional active agents are included as embodiments.Similarly methods and pharmaceutical combinations in which ribavirin isan additional active agent are provided herein.

Methods of inhibiting HCV replication in vivo comprising providing acompound or pharmaceutically acceptable salt of Formula I to a patientinfected with HCV, a concentration of the compound or salt of Formula Isufficient to inhibit HCV replicon replication in vitro are includedherein. In this instance the concentration includes an in vivoconcentration, such as a blood or plasma concentration. Theconcentration of compound sufficient to inhibit HCV replicon replicationin vitro may be determined from an assay of replicon replication such asthe assay provided in Example 9, herein.

Methods of treatment include providing certain dosage amounts of acompound or pharmaceutically acceptable salt of Formula I to a patient.Dosage levels of each active agent of from about 0.1 mg to about 140 mgper kilogram of body weight per day are useful in the treatment of theabove-indicated conditions (about 0.5 mg to about 7 g per patient perday). The amount of active ingredient that may be combined with thecarrier materials to produce a single unit dosage form will varydepending upon the patient treated and the particular mode ofadministration. In certain embodiments about 0.1 mg to about 2000 mg,from about 10 mg to about 1500 mg, from about 100 mg to about 1000 mg,from about 200 mg to about 800 mg, or from about 300 to about 600 mg ofa compound of Formula I and optionally from about 0.1 mg to about 2000mg, from about 10 mg to about 1500 mg, from about 100 mg to about 1000mg, from about 200 mg to about 800 mg, or from about 300 to about 600 mgof a compound of an additional active agent, for example an NS3aprotease inhibitor such as a compound of Formula II are provided dailyto a patient. It is preferred that each unit dosage form contains lessthan 1200 mg of active agent in total. Frequency of dosage may also varydepending on the compound used and the particular disease treated.However, for treatment of most infectious disorders, a dosage regimen of4 times daily or less is preferred and a dosage regimen of 1 or 2 timesdaily is particularly preferred.

It will be understood, however, that the specific dose level for anyparticular patient will depend upon a variety of factors including theactivity of the specific compound employed, the age, body weight,general health, sex, diet, time of administration, route ofadministration, and rate of excretion, drug combination and the severityof the particular disease in the patient undergoing therapy.

Packaged Formulations

Methods comprising providing a compound or salt of Formula I in acontainer together with instructions for using the compound to treat apatient suffering from Hepatitis C infection are included herein.

Packaged pharmaceutical compositions/combinations are also includedherein. Such packaged combinations include a compound of Formula I in acontainer together with instructions for using the combination to treator prevent a viral infection, such as a hepatitis C infection, in apatient.

The packaged pharmaceutical composition/combination may include one ormore additional active agents. In certain embodiments the additionalactive agent is an NS3a protease inhibitor, such as a compound ofFormula II.

The packaged pharmaceutical combination may include a compound orpharmaceutically acceptable salt of Formula I and the additional activeagent provided simultaneously in a single dosage form, concomitantly inseparate dosage forms, or provided in separate dosage forms foradministration separated by some amount of time that is within the timein which both the compound of Formula I and the additional active agentare within the bloodstream of the patient.

The packaged pharmaceutical combination may include a compound orpharmaceutically acceptable salt of Formula I provided in a containerwith an additional active agent provided in the same or separatecontainer, with instructions for using the combination to treat an HCVinfection in a patient.

EXAMPLES Abbreviations

The following abbreviations are used in the reaction schemes andsynthetic examples, which follow. This list in not meant to be anall-inclusive list of abbreviations used in the application asadditional standard abbreviations, which are readily understood by thoseskilled in the art of organic synthesis, may also be used in thesynthetic schemes and examples.

Ac acetyl

ACN acetonitrile

aq. aqueous

BOC t-butoxycarbonyl

DCM dichloromethane

DIEA N,N-diisopropylethylamine

DIPEA N,N-diisopropylethylamine

DME 1,2-dimethoxyethane

DMF N,N-dimethylformamide

dppf 1,1′-bis(diphenylphosphino)ferrocene

EDCI N-(3-Dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride

Et ethyl

Et₂O diethyl ether

FCC flash column chromatography

HATU O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate

MTBE methyl tert-butyl ether

PTLC preparative thin layer chromatography

rt room temperature

TEA triethylamine

TFA trifluoroacetic acid

THF tetrahydrofuran

TPP triphenylphosphine

General Considerations

All nonaqueous reactions were performed under an atmosphere of dry argongas using oven-dried glassware and anhydrous solvents. The progress ofreactions and the purity of target compounds were determined using oneof the following two HPLC methods: (1) Waters AQUITY UPLC BEH C18 1.7 μm2.1×50 mm column with an isocratic elution of 0.24 min at 90:10water:acetonitrile containing 0.05% formic acid followed by a 4.26-minlinear gradient elution from 90:10 to 10:90 at a flow rate of 1.0 mL/minwith UV (PDA), ELS, and MS (SQ in APCI mode) detection (method 1); and(2) Waters AQUITY UPLC BEH C18 1.7 μm 2.1×50 mm column with an isocraticelution of 0.31 min at 95:5 water:acetonitrile containing 0.05% formicacid followed by a 17.47-min linear gradient elution from 95:5 to 5:95at a flow rate of 0.4 mL/min with UV (PDA), ELS, and MS (SQ in APCImode) detection (method 2).

Target compounds were purified via preparative reverse-phase HPLC usinga YMC Pack Pro C18 5 μm 150×20 mm column with an isocratic elution of0.35 min at 95:5 water:acetonitrile containing 0.1% trifluoroacetic acidfollowed by a 23.3-min linear gradient elution from 95:5 to 5:95 at aflow rate of 18.9 mL/min with UV and mass-based fraction collection.

Example 1 Synthesis of Compound 10

Compound 10 was prepared via bromination of [2.2]paracyclophane asoutlined previously (Reich, H. J.; Cram, D. J. J. Am. Chem. Soc. 1969,91, 3527-3533; Reich, H. J.; Cram, D. J. J. Am. Chem. Soc. 1969, 91,3534-3543). Compounds 1, 2, 6, 8, and 10 can be obtained from commercialsources. Compounds 3-7 and 9 were prepared using general syntheticmethods known in the art.

Example 2 Synthesis of Compound 11

A deoxygenated (argon) mixture of 9 (284.2 mg), 10 (52.3 mg), K₃PO₄(248.1 mg), and PdCl₂dppf.CH₂Cl₂ (7.4 mg) in dioxane/water (5.5. mL/0.55mL) was irradiated in a microwave for 2 h at 80° C. The resultingmixture was evaporated under reduced pressure and the remaining solidwas extracted with DCM. This crude material was purified by PTLC (20cm×20 cm×2000 μm glass plates; eluted with 45:50:5 v/v/v DCM:EtOAc:MeOH,R_(f) 0.28) to give 75.3 mg of 11. The purity of 11 was determined viaanalytical reverse-phase HPLC using a 3.5-min gradient elution ofincreasing concentrations of ACN in water (10-90%) containing 0.05%formic acid with a flow rate of 1.0 mL/min on a Waters AQUITY UPLC BEHC18 1.7 μm 2.1×50 mm column with UV (PDA), ELS, and MS (SQ in APCI mode)detection. HPLC: t_(R) 1.57 min (98% purity). MS m/z calculated forC₅₆H₆₄N₈O₆ ([M]+), 945. found, 946 ([M+1]+).

Example 3 Synthesis of Dimethyl((2S,2′S)-((2S,2′S,3aS,3a′S,7aS,7a′S)-2,2′-(5,5′-(tricyclo[8.2.2.2^(4,7)]hexadeca-4,6,10,12,13,15-hexaene-5,11-diyl)bis(1H-benzo[d]imidazole-5,2-diyl))bis(octahydro-1H-indole-2,1-diyl))bis(3-methyl-1-oxobutane-2,1-diyl))dicarbamate(20)

Step 1

To a stirred solution of (2R,3aS,7aS)-octahydro-1H-indole-2-carboxylicacid (250 g, 1.0 equiv) (12) in THF (3 L) and water (1.5 L) at 0° C.,was added dropwise a cooled aq solution of 2.5 M NaOH (1 L). Thereaction mixture was stirred for 15 min at the same temperature. Thendi-tert-butyl dicarbonate (1.3 equiv) was added dropwise, maintainingthe temperature at 0° C. The resulting reaction mixture was stirred atrt for 12 h. The reaction mixture was washed with MTBE (3 times). The aqphase was acidified with 1 M aq citric acid and extracted with ethylacetate (3 times). The combined organic layers were dried over sodiumsulphate and concentrated to dryness to give(2R,3aS,7aS)-1-(tert-butoxycarbonyl)octahydro-1H-indole-2-carboxylicacid (368 g) (13).

Step 2

To a stirred solution of 4-bromo-1,2-diaminobenzene (23.1 g, 1.2 equiv),(2R,3aS,7aS)-1-(tert-butoxycarbonyl)octahydro-1H-indole-2-carboxylicacid (26.9 g, 1.0 equiv) 13, and EDCI (23.6 g, 1.2 equiv) in ACN (600mL) at 0° C. was added DIEA (21.5 mL, 1.3 equiv) dropwise. The reactionmixture was stirred for additional 1 h after completion of addition.Water (1.2 L) was added and the reaction mixture was stirred overnight.The solid powder (14) was collected, washed with water, and dried foruse in the next step without further purification (39.1 g).

Step 3

An isomeric mixture (14) of (2S,3aS,7aS)-tert-butyl2-((2-amino-4-bromophenyl)carbamoyl)octahydro-1H-indole-1-carboxylateand (2S,3aS,7aS)-tert-butyl2-((2-amino-5-bromophenyl)carbamoyl)octahydro-1H-indole-1-carboxylate(160 g, 0.36 mol) was dissolved in acetic acid (480 mL) and the reactionmixture was stirred at 65° C. until the starting materials were consumed(as judged by LC-MS analysis). The reaction was cooled to rt and thesolvent was removed under vacuum. The remaining residue was dissolved inethyl acetate (500 mL) and aq ammonia (100 mL) was added carefully.Additional water (100 mL) was added and the organic layer was separatedand collected. The aq phase was extracted with ethyl acetate (2×300 mL).The combined organic phase was washed with water (200 mL), followed bybrine (200 mL), and dried over MgSO₄. The solution was concentrated andthe remaining residue was purified by silica gel column chromatography(hexanes/ethyl acetate) to afford (2S,3aS,7 aS)-tert-butyl2-(6-bromo-1H-benzo[d]imidazol-2-yl)octahydro-1H-indole-1-carboxylate(15) (140 g).

Step 4

Under an atmosphere of argon, a mixture of (2S,3aS,7aS)-tert-butyl2-(6-bromo-1H-benzo[d]imidazol-2-yl)octahydro-1H-indole-1-carboxylate(15) (30 g, 1.0 equiv), bis(pinacolato)diborane (27.2 g, 1.5 equiv),potassium acetate (21 g, 3.0 equiv), and Pd(dppf)Cl₂ (5.7 g, 0.098equiv) in anhydrous 1,4-dioxane (300 mL) was heated at 80-90° C. for ˜4h (until the reaction was complete as judged by LC-MS). The cooled (rt)reaction mixture was diluted with ethyl acetate (300 mL), stirred withactivated carbon (60 g) for 1 h, and filtered through a pad of Celite.The filtrate was concentrated under reduce pressure and the resultingbrown foam was purified by silica gel column chromatography(hexanes/ethyl acetate, 5:1→1:2 v/v) to give (2S,3 aS,7aS)-tert-butyl2-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazol-2-yl)octahydro-1H-indole-1-carboxylateas an off-white solid) (16).

Step 5

A mixture of Br₂ (82.8 g) and iron powder (4.6 g) in DCM (1.6 L) wasstirred at rt for 1 h. To this mixture was added, in one portion, aslurry of [2.2]paracyclophane (200 g, 1.0 equiv) in DCM. The resultingmixture was heated to reflux, and to it was added slowly a solution ofBr₂ (228 g) in DCM (400 mL) over a 3-h period. After this addition wascomplete, the reaction mixture continued to reflux for 3 h, was allowedto cool to rt with stirring overnight, was washed with 5% w/v aq Na₂S₂O₃(2 L) and water (2 L), dried (MgSO₄), and evaporated to dryness. Theisolated crude solid was dissolved in hot toluene (1.2 L, ˜100° C.),allowed to cool slowly overnight to rt with stirring, and further cooledto 5° C. for 3 h. The resulting solid was collected and washed with coldtoluene (˜100 mL) to afford 4,16-dibromo[2.2]paracyclophane (17) (83 g).¹H NMR (300 MHz, CDCl₃, rt): δ 2.79-3.00 (m, 4H), 3.10-3.21 (m, 2H),3.44-3.54 (m, 2H), 6.44 (d, J=8.0 Hz, 2H), 6.51 (d, J=2.0 Hz, 2H), 7.14(dd, J=8.0 Hz, 2.0 Hz, 2H).

Step 6

Under an atmosphere of argon, a mixture of4,16-dibromo[2.2]paracyclophane (17) (20 g, 1.0 equiv),(2S,3aS,7aS)-tert-butyl2-(6-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazol-2-yl)octahydro-1H-indole-1-carboxylate(64 g, 2.5 equiv), Cs₂CO₃ (16) (44.5 g, 2.5 equiv), Pd(PPh₃)₄ (3.16 g,0.05 equiv), DMF (500 mL), and water (25 mL) was heated at 130° C. for˜2-3 h (until the reaction was complete as judged by LC-MS). Thereaction mixture was allowed to cool to rt and filtered through a pad ofsilica gel (30 g) layered with Celite. This pad was washed with DMF(2×50 mL) and the combined filtrates were added to stirred water (2.5 L)to give a pale yellow precipitate. This solid was collected byfiltration, washed with water (1 L) and ACN (500 mL), and dissolved in amixture of DCM (250 mL) and MeOH (25 mL). To this solution was added ACN(250 mL) to generate a fine slurry, which was then concentrated underreduce pressure at 30-35° C. to remove ˜150 mL of solvent. Anotherportion of ACN (500 mL) was added and additional solvent (˜100 mL) wasremoved under reduce pressure at 40-45° C. The solid was collected byfiltration and dried under vacuum to give(2S,2′S,3aS,3a′S,7aS,7a′S)-di-tert-butyl2,2′-(5,5′-(tricyclo[8.2.2.2^(4,7)]hexadeca-4,6,10,12,13,15-hexaene-5,11-diyl)bis(1H-benzo[d]imidazole-5,2-diyl))bis(octahydro-1H-indole-1-carboxylate)as a pale yellow powder (33.8 g).

Step 7

To a cooled (0° C.) solution of (2S,2′S,3aS,3a′S,7aS,7a′S)-di-tert-butyl2,2′-(5,5′-(tricyclo[8.2.2.2^(4,7)]hexadeca-4,6,10,12,13,15-hexaene-5,11-diyl)bis(1H-benzo[d]imidazole-5,2-diyl))bis(octahydro-1H-indole-1-carboxylate)(18) (20.33 g, 1.0 equiv) in DCM/MeOH (4/1 v/v, 200 mL) was added a 4 NHCl/dioxane solution (100 mL). The reaction mixture was stirred at rtfor 30 min and concentrated under reduce pressure to give a pale yellowpowder (19) (21.7 g). The solid obtained was dried under vacuum untilresidual MeOH was undetectable by ¹H NMR spectroscopic analysis. Thisthoroughly dried material was used directly in the next step.

Step 8

To a mixture of (S)-2-((methoxycarbonyl)amino)-3-methylbutanoic acid (10g, 2.3 equiv), HOBt monohydrate (8.8 g, 2.3 equiv), and ACN (50 mL) atrt was added EDCI (11.14 g, 2.3 equiv). After stirring 5 min, thisactivated acid mixture was added to a solution of the hydrochloride salt(19) from above (21.7 g) and DIEA (32 mL, 7.2 equiv) in DMF (250 mL).The reaction mixture was stirred at rt until the reaction was judged ascomplete by LC-MS analysis (˜4 h) and then poured into water (1.2 L)with stirring. The precipitate was collected by filtration, stirred inACN/water (4:1 v/v, 500 mL) overnight, collected again by filtration,and dried in vacuo to give dimethyl((2S,2′S)-((2S,2′S,3aS,3a′S,7aS,7a′S)-2,2′-(5,5′-(tricyclo[8.2.2.2⁴′⁷]hexadeca-4,6,10,12,13,15-hexaene-5,11-diyl)bis(1H-benzo[d]imidazole-5,2-diyl))bis(octahydro-1H-indole-2,1-diyl))bis(3-methyl-1-oxobutane-2,1-diyl))dicarbamate(20) (22.62 g).

¹H NMR (300 MHz, DMSO-d₆, 120° C.): δ0.87 (d, J=6.5 Hz, 6H), 0.92 (d,J=6.5 Hz, 6H), 1.20-1.60 (m, 6H), 1.65-2.10 (m, 12H), 2.31 (m, 2H),2.38-2.52 (m, 2H), 2.54-2.76 (m, 4H), 2.85 (m, 2H), 3.07 (m, 2H), 3.45(m, 2H), 3.57 (br s, 6H), 4.07 (t, J=8.0 Hz, 2H), 4.34 (m, 2H), 5.28 (t,J=8.5 Hz, 2H), 6.51 (br, 2H), 6.57 (d, J=8.0 Hz, 2H), 6.73 (s, 2H), 6.76(d, J=8.0 Hz, 2H), 7.33 (d, J=8.5 Hz, 2H), 7.64 (d, J=8.5 Hz, 2H), 7.72(s, 2H).

Example 4 Synthesis of Dimethyl((2S,2′S,3R,3′R)-((2S,2′S,3aS,3a′S,7aS,7a′S)-2,2′-(5,5′-(tricyclo[8.2.2.2^(4,7)]hexadeca-4,6,10,12,13,15-hexaene-5,11-diyl)bis(1H-benzo[d]imidazole-5,2-diyl))bis(octahydro-1H-indole-2,1-diyl))bis(3-methoxy-1-oxobutane-2,1-diyl))dicarbamate(22)

Step 1

O-Methyl-L-threonine (25 g, 0.19 mol) was dissolved in 1,4-dioxane (125mL) and cooled to 0° C. An aq 2 M NaOH (22.5 g, 0.56 mol, 3 equiv)solution was then added to the reaction mixture followed by methylchloroformate (17.4 mL, 0.22 mol, 1.2 equiv) at the same temperature.The reaction mixture was warmed to rt and stirred for 16 h. The reactionmixture was washed with ethyl acetate (500 mL). The aq layer wasacidified with 3 N HCl (up to pH 2) and extracted with ethyl acetate(3×250 mL). The combined organic layers were dried over Na₂SO₄ andconcentrated to afford the crude product (23 g). Ethyl acetate (46 mL)was added to the crude product and heated at 80° C. to obtain a clearsolution. This solution was then cooled to 0° C. The solid obtained wasfiltered and dried to afford the desired pure product (21) (18.75 g).

Step 2

DIEA (32.8 mL, 0.192 mol) was added dropwise to a mixture of thehydrochloride salt from above (32.0 g, 0.0384 mol),Moc-O-methyl-L-threonine (18.3 g, 0.0960 mol), and HATU (36.5 g, 0.09604mol) in DMF (160 mL) at 0° C. The reaction mixture was allowed to warmto rt, stirred for 16 h, and poured into water (1.6 L). The resultingsolid was collected by filtration and dissolved in DCM (500 mL). Thissolution was washed with water (100 mL), washed with brine (50 mL),dried over sodium sulphate, and concentrated under reduced pressure. Theresidue was purified by silica gel column chromatography to givedimethyl((2S,2′S,3R,3′R)-((2S,2′S,3aS,3a′S,7aS,7a′S)-2,2′-(5,5′-(tricyclo[8.2.2.2⁴′⁷]hexadeca-4,6,10,12,13,15-hexaene-5,11-diyl)bis(1H-benzo[d]imidazole-5,2-diyl))bis(octahydro-1H-indole-2,1-diyl))bis(3-methoxy-1-oxobutane-2,1-diyl))dicarbamate(22) (30 g). ¹H NMR (400 MHz, DMSO-d₆, rt): δ0.91-1.04 (m, 6H),1.21-1.59 (m, 6H), 1.64-1.88 (m, 6H), 1.90-2.07 (m, 4H), 2.23-2.49 (m,6H), 2.62 (m, 2H), 2.85 (m, 2H), 3.08 (m, 2H), 3.19 (s, 3H), 3.20 (s,3H), 3.42 (m, 2H), 3.57 (s, 6H), 4.15 (apparent t, J=8.0 Hz, 2H), 4.47(m, 2H), 5.16 (apparent t, J=8.5 Hz, 2H), 6.52 (apparent t, J=8.5 Hz,2H), 6.75 (s, 2H), 6.82 (m, 2H), 7.41 (m, 2H), 7.71 (apparent t, J=6.0Hz, 2H), 7.64-7.79 (m, 4H).

Example 5 Synthesis of Dimethyl((2S,2′S,3R,3′R)-((2S,2′S,3aS,3a′S,7aS,7a′S)-2,2′-(5,5′-(tricyclo[8.2.2.2^(4,7)]hexadeca-4,6,10,12,13,15-hexaene-5,11-diyl)bis(1H-benzo[d]imidazole-5,2-diyl))bis(octahydro-1H-indole-2,1-diyl))bis(3-methoxy-1-oxobutane-2,1-diyl))dicarbamate(23)

Dimethyl((2S,2′S,3R,3′R)-((2S,2′S,3aS,3a′S,7aS,7a′S)-2,2′-(5,5′-(tricyclo[8.2.2.2^(4,7)]hexadeca-4,6,10,12,13,15-hexaene-5,11-diyl)bis(1H-benzo[d]imidazole-5,2-diyl))bis(octahydro-1H-indole-2,1-diyl))bis(3-hydroxy-1-oxobutane-2,1-diyl))dicarbamatewas prepared in a manner analogous to that described above for thesynthesis of dimethyl((2S,2′S,3R,3′R)-((2S,2′S,3aS,3a′S,7aS,7a′S)-2,2′-(5,5′-(tricyclo[8.2.2.2⁴′⁷]hexadeca-4,6,10,12,13,15-hexaene-5,11-diyl)bis(1H-benzo[d]imidazole-5,2-diyl))bis(octahydro-1H-indole-2,1-diyl))bis(3-methoxy-1-oxobutane-2,1-diyl))dicarbamate.¹H NMR (400 MHz, DMSO-d₆, rt): ¹H NMR (400 MHz, DMSO-d₆, rt): δ0.95-1.04(m, 6H), 1.20-1.56 (m, 6H), 1.64-1.85 (m, 6H), 1.90-2.09 (m, 4H), 2.25(m, 2H), 2.32-2.49 (m, 4H), 2.63 (m, 2H), 2.83 (m, 2H), 3.06 (m, 2H),3.39 (m, 2H), 3.57 (s, 6H), 3.69 (m, 2H), 4.06 (apparent t, J=7.5 Hz,2H), 4.46 (m, 2H), 4.72 (m, 2H), 5.13 (m, 2H), 6.44-6.57 (m, 2H),6.68-6.87 (m, 4H), 7.23-7.35 (m, 4H), 7.53-7.78 (m, 4H).

Example 6 Synthesis of Compound 29 Via the Tetraamine Synthetic Route

Step 1

1,2-Diamino-4-bromobenzene (10 g, 0.053 mol) was dissolved in DCM (150mL) and cooled to 0° C. A solution of NaOH (50 mL, 2.5 mol) was addeddropwise at the same temperature. After 15 min, di-tert-butyldicarbonate (58 g, 0.26 mol) was added dropwise at the same temperature.Then the reaction mixture was allowed to warm to room temperature,stirred for 16 h, diluted with DCM (100 mL), and washed with water (100mL). The organic layer was separated, dried over Na₂SO₄, andconcentrated. The crude material was purified by silica gel columnchromatography (petroleum ether/ethyl acetate, 1:1 v/v) to afford thedesired product (18 g)

Step 2

Under an atmosphere of argon, a mixture of di-tert-butyl(4-bromo-1,2-phenylene)dicarbamate (24) (18 g, 0.046 mol),bis(pinacolato)diboron (17.7 g, 0.070 mol), potassium acetate (13.66 g,0.14 mol), and Pd(dppf)Cl₂ (3.8 g, 0.0046 mol) in 1,4-dioxane (360 mL)was heated at 85° C. for 16 h. The reaction mixture was then dilutedwith ethyl acetate (200 mL) and filtered through a bed of Celite. Thefiltrate was concentrated under reduced pressure and the remainingmaterial was purified by silica gel column chromatography (petroleumether/ethyl acetate, 80:20 v/v) to afford the desired product (25) (16.0g).

Step 3

Under an atmosphere of argon, a mixture of4,16-dibromo[2.2]paracyclophane (17) (6 g, 0.014 mol), di-tert-butyl(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1,2-phenylene)dicarbamate(17.78 g, 0.032 mol), aq Cs₂CO₃ solution (15.98 g, 0.049 mol, in 66 mLwater), and Pd(PPh₃)₄ (1.33 g, 0.0016 mol) was heated in a sealed tubeat 80° C. for 16 h. The reaction mixture was poured into water (250 mL)and the resulting precipitate was collected by filtration and washedwith water. This crude material was purified by column chromatography(petroleum ether/ethyl acetate, 6:4 v/v) to afford the desired product(26) (5.0 g).

Step 4

The tetra Boc-protected product from above (26) (10 g) was added to TFA(100 mL) at 0-5° C. After completion of the addition, the reactionmixture was warmed to rt and stirred for 3 h. The reaction mixture wasthen concentrated and co-evaporated with DCM (3×50 mL). The crudematerial (27) was used directly in the next step.

Step 5

DIEA (1 mL, 0.0125 mol) was added dropwise to a cooled (0° C.) mixtureof (S)-5-(tert-butoxycarbonyl)-5-azaspiro[2.4]heptane-6-carboxylic acid(0.5 g, 0.00057 mol), HOBt (0.35 g, 0.0026 mol), EDCI (0.5 g, 0.0026mol), and the TFA salt (27) from above (0.343 g, 0.0014 mol) in DMF (5mL) at 0° C. After the addition was complete, the reaction mixture wasallowed to warm to rt and stirred for 16 h. The reaction mixture waspoured into water and the precipitate (28) was collected by filtrationand purified by silica gel column chromatography to give the desiredproduct (0.32 g).

Step 6

Acetic acid (5 mL) was added to the diamide product from above (28)(0.32 g, 0.00035 mol) and heated at 45° C. for 4 h. The reaction mixturewas evaporated and the residue was diluted with ethyl acetate (95 mL),then washed with aq NaHCO₃ (2×25 mL) and water (2×30 mL). The organiclayer was separated, dried over Na₂SO₄, and evaporated under reducedpressure. The crude material was purified by column chromatography togive the desired product (29) (0.2 g).

Step 7

The boc-protected product from above (0.09 g) was dissolved in DCM (0.9mL) and cooled at 0° C. Then 4 N HCl/dioxane (0.9 mL) was addeddropwise. The reaction mixture was warmed to rt and stirred for 3 h.Then the volatiles were removed under vacuum and co-evaporated with DCM(3×50 mL). The remaining crude material (30) was used directly in thenext step without further purification.

Step 8

The hydrochloride salt (30) from above (0.011 g, 0.0000141 mol, 1.0equiv) was dissolved in DMF (1 mL) and cooled to 0° C. To this cooledsolution were added (S)-2-((methoxycarbonyl)amino)-3-methylbutanoic acid(0.0062 g, 0.000033 mol, 2.5 equiv), HOBt (0.0044 g, 0.000033 mol, 2.5equiv) and EDCI (0.0063 g, 0.000033 mol, 2.5 equiv). DIEA (0.02 mL,0.00013 mol, 10 equiv) was then added dropwise at the same temperature.The reaction mixture was allowed to warm to rt and stirred for 16 h. Thereaction mixture was then poured into water (25 mL) and the precipitatedsolid was collected by filtration, dried, and purified by silica gelcolumn chromatography to give the desired product (31) (3 mg). ¹H NMR(400 MHz, DMSO-d₆, rt): δ0.91-1.04 (m, 6H), 1.21-1.59 (m, 6H), 1.64-1.88(m, 6H), 1.90-2.07 (m, 4H), 2.23-2.49 (m, 6H), 2.62 (m, 2H), 2.85 (m,2H), 3.08 (m, 2H), 3.19 (s, 3H), 3.20 (s, 3H), 3.42 (m, 2H), 3.57 (s,6H), 4.15 (apparent t, J=8.0 Hz, 2H), 4.47 (m, 2H), 5.16 (apparent t,J=8.5 Hz, 2H), 6.52 (apparent t, J=8.5 Hz, 2H), 6.75 (s, 2H), 6.82 (m,2H), 7.41 (m, 2H), 7.71 (apparent t, J=6.0 Hz, 2H), 7.64-7.79 (m, 4H).

Example 7 Synthesis of Ferrocene NS5A Inhibitors (32)

Ferrocene compounds are prepared by the method discussed in Butler, I.R., et al., “A Convenient Preparation of Iodoferrocenes,” Polyhedron(1993) 12: 129-131. To a stirred solution of 1,1′-diiodoferrocene (220mg, 0.5 mmol) in 1,4-dioxane (10 mL) was added methyl((S)-3-methyl-1-oxo-1-((2S,3aS,7aS)-2-(5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-benzo[d]imidazol-2-yl)octahydro-1H-indol-1-yl)butan-2-yl)carbamate(1.1 g, 4 equiv), K₃PO₄ (853 mg, 2 M aq solution, 8 equiv), andPdCl₂dppf (49 mg, 12 mol %) under an atmosphere of argon. The resultingmixture was subjected to microwave irradiation (CEM Discover System) at80° C. for 1 h. The reaction mixture was allowed to cool to roomtemperature, filtered, and concentrated in vacuo. The remaining residuewas purified by preparative HPLC to give the desired product as thetrifluoroacetate salt (43 mg). ¹H NMR (400 MHz, DMSO-d₆, 27° C.): δ 0.63(d, J=6.5 Hz, 6H), 0.78 (d, J=6.5 Hz, 6H), 1.13-1.49 (m, 6H), 1.55-1.87(m, 10H), 1.95 (m, 2H), 2.26 (m, 2H), 2.35 (m, 2H), 2.42 (m, 2H), 3.48(s, 6H), 3.83 (t, J=8.0 Hz, 2H), 4.18 (m, 4H), 4.37 (m, 2H), 4.67 (m,4H), 5.10 (dd, J=10.0 Hz, 7.5 Hz, 2H), 7.39 (d, J=9.0 Hz, 2H), 7.42 (d,J=9.0 Hz, 2H), 7.45 (d, J=8.0 Hz, 2H), 7.53 (s, 2H).

Example 8 Additional Compounds of Formula I

The following compounds are prepared by the methods set forth inExamples 1-6.

EC50 (μM) HPLC No. Structure NS5A1b NS5A1b LC method MS 40

.000012 1.57 1 945 41

<0.0032 0.00781 1.67 1 923 42

<0.0032 0.0044 2.15 1 945 43

0.00285 2.86 1 1001 44

0.00408 1.35 1 805 dimethyl((2S,2′S)-((2S,2′S)-2,2′-(5,5′-(bicyclo[1.1.1]pentane-1,3-diylbis(4,1-phenylene))bis(1H-imidazole-5,2-diyl))bis(pyrrolidine-2,1-diyl))bis(3-methyl-1-oxobutane-2,1-diyl))dicarbamate 45

<0.0032 1.93 1 893 46

<0.00317 1.69 1 870 47

0.00502 1.67 1 870 48

<0.00317 6.06 2 871 49

0.0102 1.94 1 962 50

0.00841 1.8 1 957 51

>1 1.62 1 889 52

0.00983 1.88 1 1014 53

0.211 1.78 1 1009 54

>1 1.64 1 942 55

>1 2.02 1 998 56

0.00164 8.18 2 979 57

>1 2.04 1 1062 58

>1 1.9 1 994 59

0.548 2.21 1 1066 60

0.128 2.45 1 1054 61

0.0752 2.43 1 1118 62

0.075 2.58 1 1122 63

0.899 2.33 1 1051 64

65

66

0.000005 1.99 1 921 67

68

69

70

2.21 1 921 71

72

2.03 1 1079 73

1.86 1 1143 74

75

76

77

1.33 1 917 78

1.64 1 981 79

80

81

82

83

84

85

86

87

0.00653 0.0184 1.87 1 930 88

0.00643 0.0618 1.91 1 993 89

90

91

1.68 1 929 92

93

94

95

1.76 1 957 96

2.11 1 1029 97

2.51 1 1060 98

2.93 1 1138 99

100

101

2.17 1 1028 102

0.0119 0.0358 2.79 1 1030 103

<0.00317 0.47 1.99 1 922 104

2.24 1 996 105

1.51 1 888 106

107

108

1.63 1 915 109

0.00883 0.0118 1.63 1 926 110

0.000006 2.11 1 1024 111

0.000005 2.37 1 1034 112

113

114

115

116

117

118

119

120

121

122

123

124

1.8 1 910 125

2.73 1 1018 126

2.08 1 1222 127

0.633 >1 8.2 2 923 128

0.000008 2.15 1 1006 129

130

0.000009 2.2 1 1261 131

132

133

134

135

136

137

138

139

140

141

142

143

144

145

146

147

148

149

150

151

152

153

154

155

156

Example 9 Assay for Identifying Compounds which Inhibit HCV Replication

Compounds claimed herein are tested for the ability to inhibit viralreplication of the Hepatitis C replicon in cultured cells in which theHCV replicon construct has been incorporated. The HCV replicon systemwas described by Bartenschlager, et. al (Science, 285, pp. 110-113(1999)). The replicon system is predictive of in vivo anti-HCV activity;compounds that are active in humans uniformly evidence activity in thereplicon assay.

In this assay HCV replicon containing cells are treated with differentconcentrations of the test compound to ascertain the ability of the testcompound to suppress replication of the HCV replicon. As a positivecontrol, HCV replicon-containing cells are treated with differentconcentrations of interferon alpha, a known inhibitor of HCVreplication. The replicon assay system includes NeomycinPhosphotransferase (NPT) as a component of the replicon itself in orderto detect the transcription of replicon gene products in the host cell.Cells in which the HCV replicon is actively replicating have high levelsof NPT; the level of NPT is proportional to HCV replication. Cells inwhich the HCV replicon is not replicating also have low levels of NPTand thus do not survive when treated with Neomycin. The NPT level ofeach sample is measured using a captured ELISA.

A protocol for testing compounds for the ability to inhibit viralreplication of the Hepatitis C replicon cultured cells in which thereplicon construct has been incorporated, follows.

9A. HCV Replicon and Replicon Expression

The HCV genome consists of a single ORF that encodes a 3000 amino acidpolyprotein. The ORF is flanked on the 5′ side by an untranslated regionthat serves as an internal ribosome entry site (IRES) and at the 3′ sideby a highly conserved sequence necessary for viral replication (3′-NTR).The structural proteins, necessary for viral infection, are located nearthe 5′ end of the ORF. The non-structural proteins, designated NS2 toNS5B comprise the remainder of the ORF.

The HCV replicon contains, 5′-3′, the HCV-IRES, the neomycinphosphotransferase (neo) gene, the IRES of encephalomyocarditis virus,which directs translation of HCV sequences NS3 to NS5B, and the 3′-NTR.The sequence of the HCV replicon has been deposited in GenBank(Accession no. AJ242652).

The replicon is transfected into Huh-7 cells using standard methods suchas electroporation.

9B. Cell Maintenance

The equipment and materials include, but are not limited to, Huh-7 HCVreplicon-containing cells, maintenance media (DMEM (Dulbecco's modifiedEagle media) supplemented with 10% FBS, L-glutamine, non-essential aminoacids, penicillin (100 units/nil), streptomycin (100 micrograms/nil),and 500 micrograms/ml of Geneticin (G418), screening media (DMEMsupplemented with 10% FBS, L-glutamine, non-essential amino acids,penicillin (100 units/nil) and streptomycin (100 micrograms/nil)), 96well tissue culture plates (flat bottom), 96 well plates (U bottom fordrug dilution), Interferon alpha for positive control, fixation reagent(such as methanol: acetone), primary antibody (rabbit anti-NPTII),secondary antibody: Eu-N11, and enhancement solution.

HCV replicon-containing cells support high levels of viral RNA repliconreplication when their density is suitable. Over-confluency causesdecreased viral RNA replication. Therefore, cells must be kept growingin log phase in the presence of 500 micrograms/ml of G418. Generally,cells should be passed twice a week at 1: 4-6 dilution. Cell maintenanceis conducted as follows:

HCV replicon-containing cells are examined under a microscope to ensurethat cells growing well. Cells are rinsed once with PBS and 2 ml trypsinis added. The cell/trypsin mixture is incubated at 37° C. in a CO₂incubator for 3-5 minutes. After incubation 10 ml of complete media isadded to stop the trypsinization reaction. Cells are blown gently, putinto a 15 ml tube, and spun at 1200 rpm for 4 minutes. Thetrypsin/medium solution is removed. Medium (5 ml) is added and the cellsare mixed carefully. The cells are counted.

The cells are then seeded onto 96-well plates at a density of 6000-7500cells/100 microliters/well (6-7.5×105 cells/10 ml/plate). The plates arethen incubated at 37° C. in a 5% CO2 incubator.

Cells are examined under a microscope approximated 24 hours afterseeding and prior to adding drugs. If counting and dilution wereperformed correctly, cells are 60-70% confluent and nearly all cellsshould attach and spread evenly in the well.

6C. Treatment of HCV-Replicon Containing Cells with Test Compound

HCV replicon-containing cells are rinsed with once PBS once; 2 mls oftrypsin are then added. Cells are incubated at 37° C. in a 5% CO2incubator for 3-5 minutes. 10 mls of complete medium is added to stopthe reaction. Cells are blown gently, put into a 15 ml tube, and spun at1200 rpm for four minutes. The trypsin/medium solution is removed and 5mls of medium (500 ml DMEM (high glucose)) from BRL catalog #12430-054;50 mls 10% FBS, 5% Geneticin G418 (50 mg/ml, BRL catalog #10131-035), 5ml MEM non-essential amino acids (100×BRL #11140-050) and 5 ml pen-strep(BRL #15140-148) is added. The cells and media are mixed carefully

Cells are plated with screening medium (500 ml DMEM (BRL #21063-029), 50ml FBS (BRL #10082-147) and 5 ml MEM non-essential amino acid (BRL#11140-050) at 6000-7500 fcells/100 μl/well of 96 well plate (6-7.5×105cells/10 ml/plate). Plates are placed into 37° C. 5% CO₂ incubatorovernight.

4D. Assay

The following morning, drugs (test compounds or interferon alpha) arediluted in 96 well U bottom plates with media or DMSO/media, dependingon the final concentration chosen for screening. Generally for 6concentrations of each test compounds ranging from 10 micromolar to 0.03micromolar are applied. 100 μl of the test compound dilution is placedin wells of the 96 well plate containing the HCV replicon cells. Mediawithout drug is added to some wells as a negative controls. DMSO isknown to affect cell growth. Therefore, if drugs diluted in DMSO areused, all wells, including negative control (media only) and positivecontrol (interferon alpha) wells, must contain the same concentration ofDMSO, for single dose screening. The plates are incubated at 37° C. in ahumidified 5% CO2 environment for three days.

On day four, the NTPII assay is quantitated. The medium is poured fromthe plates and the plates are washed once in 200 μl of PBS. The PBS isthen decanted and the plates tapped in a paper towel to remove anyremaining PBS. Cells are fixed in situ with 100 μl/well of pre-cooled(−20° C.) methanol:acetone (1:1) and the plates are placed at −20° C.for 30 minutes.

The fixing solution is poured from the plates and the plates allowed toair-dry completely (approximately one hour). The appearance of the driedcell layer is recorded and the density of the cells in the toxic wellsis scored with the naked eye. Alternatively cell viability may beassessed using the MTS assay described below.

The wells are blocked with 200 μl of blocking solution (10% FBS; 3% NGSin PBS) for 30 minutes at room temperature. The blocking solution isremoved and 100 μl of rabbit anti-NPTII diluted 1:1000 in blockingsolution is added to each well. The plates are then incubated 45-60minutes at room temperature. After incubation, wells are washed sixtimes with PBS-0.05% Tween-20 solution. 100 μl of 1:15,000 dilutedEuropium (EU)-conjugated goat anti-rabbit in blocking buffer is added toeach well and incubated at room temperature for 30-45 minutes. Theplates are washed again and 100 μl of enhancement solution (Perkin Elmer#4001-0010) is added to each well. Each plate is shaken (approx. 30 rpm)in a plate shaker for three minutes. 95 μl is transferred from each wellto a black plate; the EU signal is quantitated in a Perkin-Elmer VICTORplate reader (EU-Lance).

When tested in this assay Compounds 11, 16, 25, 33, 38, 39, and 40exhibit EC50 values of about 10 micromolar or less.

Example 10 Cytotoxicity Assays

To insure that the decrease in replicon replication is due to compoundactivity against the HCV replicon rather than nonspecific toxicityassays are used to quantitate compound cytotoxicity.

10A. Cellular Protein Albumin Assay for Cytotoxicity

Cellular protein albumin measurements provide one marker ofcytotoxicity. The protein levels obtained from cellular albumin assaysmay also be used to provide a normalization reference for antiviralactivity of compounds. In the protein albumin assay HCVreplicon-containing cells are treated for three days with differentconcentrations of helioxanthin; a compound that is known to be cytotoxicat high concentrations. The cells are lysed and the cell lysate used tobind plate-bound goat anti-albumin antibody at room temperature (25° C.to 28° C.) for 3 hours. The plate is then washed 6 times with 1×PBS.After washing away the unbound proteins, mouse monoclonal anti-humanserum albumin is applied to bind the albumin on the plate. The complexis then detected using phosphatase-labeled anti-mouse IgG as a secondantibody.

10B. MTS Assay for Cytotoxicity

Cell viability may also be determined by CELLTITER 96 AQUEOUS ONESolution Cell Proliferation Assay (Promega, Madison Wis.), acolorimetric assay for determining the number of viable cells. In thismethod, before fixing the cells, 10-20 μl MTS reagent is added to eachwell according to manufacturer's instructions, plates are incubated at37° C. and read at OD 490 nm. During the incubation period living cellscovert the MTS reagent to a formazan product which absorbs at 490 nm.Thus the 490 nm absorbance is directly proportional to the number ofliving cells in culture.

A direct comparison of the Cellular Albumin and MTS methods fordetermining cytotoxicity may be obtained as follows: Cells are treatedwith different concentrations of test compound or Helioxanthin for athree day-period. Prior to lysis for detection albumin as describedabove, the MTS reagent is added according to manufacturer's instructionto each well and incubate at 37° C. and read at OD 490 nm. The cellularalbumin quantitation is then performed as described above.

What is claimed is:
 1. A compound of the formula:


2. A pharmaceutical composition comprising an effective amount of acompound of the formula:

to treat HCV in a patient diagnosed with HCV or its pharmaceuticallyacceptable salt, further comprising an additional active agent, in apharmaceutically acceptable carrier.
 3. The pharmaceutical compositionof claim 2, wherein the additional active agent is a protease inhibitor.4. The pharmaceutical composition of claim 2, wherein the additionalactive agent is a nucleoside analog.
 5. The pharmaceutical compositionof claim 2, wherein the additional active agent is a NS5a inhibitor. 6.The pharmaceutical composition of claim 2, wherein the additional activeagent is a NS5b inhibitor.
 7. The pharmaceutical composition of claim 2,wherein the additional active agent is a NS4a inhibitor.
 8. Thepharmaceutical composition of claim 2, wherein the additional activeagent is a NS4b inhibitor.
 9. The pharmaceutical composition of claim 2,wherein the additional active agent is a polymerase inhibitor.
 10. Thepharmaceutical composition of claim 2, wherein the additional activeagent is a NS3a inhibitor.
 11. The pharmaceutical composition of claim2, wherein the additional active agent is an immunomodulatory compound.12. The pharmaceutical composition of claim 2, in an oral dosage form.13. The pharmaceutical composition of claim 3, in an oral dosage form.14. The pharmaceutical composition of claim 4, in an oral dosage form.15. The pharmaceutical composition of claim 5, in an oral dosage form.16. The pharmaceutical composition of claim 6, in an oral dosage form.17. The pharmaceutical composition of claim 7, in an oral dosage form.18. The pharmaceutical composition of claim 8, in an oral dosage form.19. The pharmaceutical composition of claim 9, in an oral dosage form.20. The pharmaceutical composition of claim 10, in an oral dosage form.21. The pharmaceutical composition of claim 11, in an oral dosage form.22. The pharmaceutical composition of claim 2, in a tablet.
 23. Thepharmaceutical composition of claim 3, in a tablet.
 24. Thepharmaceutical composition of claim 4, in a tablet.
 25. Thepharmaceutical composition of claim 5, in a tablet.
 26. Thepharmaceutical composition of claim 6, in a tablet.
 27. Thepharmaceutical composition of claim 7, in a tablet.
 28. Thepharmaceutical composition of claim 8, in a tablet.
 29. Thepharmaceutical composition of claim 9, in a tablet.
 30. Thepharmaceutical composition of claim 10, in a tablet.
 31. Thepharmaceutical composition of claim 11, in a tablet.
 32. A method totreat HCV in a patient diagnosed with HCV with a pharmaceuticalcomposition comprising a compound of the formula:

or its pharmaceutically acceptable salt, and further comprising anadditional active agent optionally in a pharmaceutically acceptablecarrier.
 33. The method of claim 32, wherein the additional active agentis a protease inhibitor.
 34. The method of claim 32, wherein theadditional active agent is a nucleoside analog.
 35. The method of claim32, wherein the additional active agent is a NS5a inhibitor.
 36. Themethod of claim 32, wherein the additional active agent is a NS5binhibitor.
 37. The method of claim 32, wherein the additional activeagent is a NS4a inhibitor.
 38. The method of claim 32, wherein theadditional active agent is a NS4b inhibitor.
 39. The method of claim 32,wherein the additional active agent is a polymerase inhibitor.
 40. Themethod of claim 32, wherein the additional active agent is a NS3ainhibitor.
 41. The method of claim 32, wherein the additional activeagent is an immunomodulatory compound.